Self-stabilizing linker conjugates

ABSTRACT

The present invention provides Ligand-Drug Conjugates, Drug-Linkers, Linkers, and Ligand-Linker Conjugates comprising a self-stabilizing linker assembly component.

CONTINUITY

This application is a continuation of U.S. patent application Ser. No.14/727,340 filed Jun. 1, 2015, which is a divisional of U.S. patentapplication Ser. No. 13/799,244 filed Mar. 13, 2013 (now U.S. Pat. No.9,504,756), which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 61/647,373, filed May15, 2012; U.S. Provisional Application Ser. No. 61/770,983, filed Feb.28, 2013; and U.S. Provisional Application Ser. No. 61/773,067, filedMar. 5, 2013, the disclosures of each being incorporated herein byreference.

BACKGROUND OF THE INVENTION

The antibody-drug conjugate (ADC) field has made significant advanceswith the FDA approval of Brentuximab Vedotin for the treatment of aselect group of patients and with the advancement of many other ADCs inthe clinic. The linker component of ADCs is one important feature indeveloping optimized therapeutic agents that are highly active at welltolerated doses. The electrophilic maleimide functional group has provenvery useful in the preparation of ADCs due to its high degree ofspecificity for reacting with thiol groups and the very fast thioladdition kinetics under gentle conditions.

As has been noted by multiple investigators in the bioconjugate field,the thio-substituted product of the reaction between the electrophilicmaleimide functional group and free thiol of antibody is subject to slowelimination, thus reversing the above reaction:

When this reversible reaction occurs in a purified preparation of theADC, the reaction is largely undetectable because the maleimide andthiol which are regenerated through the elimination process simply reactagain, thus reforming the intact conjugate. However, when other thiolsare present, the net effect can be the transfer of the maleimide fromthe antibody of the ADC onto any other available thiol. This process hasbeen documented to occur in plasma, in which the maleimide of an ADCtransfers to cysteine 34 of serum albumin (Alley et al., BioconjugateChem. 2008, 19, 759-765). This process has also been reported when anADC is incubated in the presence of excess cysteine or glutathione(Jununtula et al., Nature Biotech, 2012). The present inventionprovides, inter alia, bioconjugates that do not undergo this transferreaction.

BRIEF SUMMARY OF THE INVENTION

The invention provides inter alia, Linkers, Drug-Linkers, Ligand-DrugConjugates, Ligand-Linker Conjugates, Ligand-Functional AgentConjugates, and Functional Agent-Linkers, and methods of preparing andusing them. The Ligand-Drug Conjugates are stable in circulation, yetcapable of inflicting cell death once released in the vicinity or withintumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a reaction scheme illustrating the reduction of theinterchain disulfides of a human IgG1, followed by conjugation of theresulting thiols with a self-stabilizing linker and subsequenthydrolysis of the succinimide ring (Top); and the use of massspectrometry to monitor the change in the molecular weight of theantibody conjugates due to hydrolysis (Bottom).

FIGS. 2A and 2B illustrate the timecourse of succinimide ring hydrolysisof a self-stabilizing antibody conjugate by electrospray massspectrometry. Conjugation of fully reduced cAC10 withmaleimido-DPR-val-cit-PAB-MMAE was performed at pH 7.2 and 22° C., thensamples were subjected to analysis by LC-MS at the indicated times (FIG.2A). The resulting data of % hydrolysis was plotted versus time and fitto an exponential equation to determine kinetic parameters (FIG. 2B).

FIG. 3 provides the hydrolysis kinetic profiles for bioconjugatesprepared with an IgG1 antibody and self-stabilizing linkers with varyingspacing between the maleimide and the basic group (a primary amine).Conjugation was performed at pH 8 and 37° C., then hydrolysis of theIgG1 light chain conjugate was immediately monitored by massspectrometry, plotted as a function of time, and fit to an exponentialequation.

FIG. 4 provides kinetic profiles of the hydrolysis of bioconjugatesprepared with an IgG1 antibody and self-stabilizing maleimide linkerswith varying spacing between the maleimide and the basic group (aprimary amine). Conjugation was performed at pH 8 and 37° C., thenhydrolysis of the IgG1 light chain conjugate was immediately monitoredby mass spectrometry, plotted as a function of time, and fit to anexponential equation.

FIG. 5 provides hydrolysis kinetic profiles for bioconjugates preparedwith an IgG1 antibody and various N-substituted maleimides. Conjugationwas performed at pH 7.4 and 22° C., then hydrolysis of the IgG1 lightchain conjugate was immediately monitored by mass spectrometry, plottedas a function of time, and fit to an exponential equation. Hydrolysis ofthe maleimido-caproyl conjugate (bottom structure) is too slow toproduce any detectable hydrolysis in 24 hours under these conditions.The presence of the carboxamide electron withdrawing group (EWG) or theprimary amine (BASE) accelerate the hydrolysis, and the combination ofthe two (top structure) results in a conjugate which hydrolyzes with ahalf-life of less than 20 minutes under these mild conditions.

FIG. 6 provides hydrolysis kinetic profiles for self-stabilizingmaleimido drug-linkers prepared with α-diaminopropionic acid (α-DPR,open circles) and with β-diaminopropionic acid (β-DPR, filled circles).Although isomers of each other, the positioning of the basic amino groupand the electron withdrawing carboxamide relative to the succinimideresults in a 17-fold difference in the rate of succinimide hydrolysis.

FIGS. 7A and 7B illustrate the change in drug loading over time for anADC prepared with a self-stabilizing maleimido-DPR drug-linker versusone prepared with a maleimido-caproyl drug linker when incubated in abuffer containing excess thiol. The reversed-phase chromatograms of thetwo ADCs at time zero and time 14 days after incubation is shown in thetop panel. Chromatographic peak assignments L0, L1, H0, H1, H2, and H3correspond to unconjugated light chain, light chain with one drug,unconjugated heavy chain, and heavy chain with 1, 2, or 3 drugs,respectively. The self-stabilizing maleimido-DPR drug-linker isrepresented with open circles versus one prepared with amaleimido-caproyl drug linker (open squares). Drug loading remainsconstant at 8 per antibody for the self-stabilizing drug-linker (opencircles), but falls to 4 drugs per antibody over 14 days for themaleimido-caproyl drug linker (open squares), reflecting loss of drug bymaleimide elimination.

FIG. 8 illustrates the change in drug loading over time for ADCsprepared with a self-stabilizing maleimido-DPR drug-linker and amaleimido-caproyl drug linker, when incubated in rat plasma at 37° C.(R=val-cit-PAB-MMAE). ADC samples at each timepoint were purified by IgSelect affinity resin and their drug loading evaluated by reversed-phaseHPLC analysis of the ADCs.

FIG. 9 provides the stability profile of drugs conjugated to antibodiesvia a maleimido-caproyl drug-linker (squares) or a self-stabilizingmaleimide linker(circles) during incubation in rat (open symbols) orhuman (filled symbols) plasma (R=val-cit-PAB-MMAE). ADCs were capturedon Protein A affinity resin at each timepoint and the drug releasedenzymatically via its protease-cleavable linker. The released drug wasthen quantified by LC-MS/MS and normalized to the initial value. Eachtimepoint reflects the percent of the conjugated drug that was observedat t0.

FIG. 10 illustrates the decrease in drug loading in vivo (rats) for ADCsprepared with a self-stabilizing maleimido-DPR drug-linker and amaleimido-caproyl drug linker (R=val-cit-PAB-MMAE). ADCs were dosed i.v.and plasma samples from each timepoint were purified by Ig Selectaffinity resin and their drug loading evaluated by reversed-phase HPLCanalysis of the ADCs.

FIG. 11 illustrates the antitumor activity of ADCs in a murine xenograftmodel of ALCL (Karpas-299 cell line). ADCs were prepared with theanti-CD30 antibody cAC10 and drug linkers containing theval-cit-PAB-MMAE cytotoxic payload linked to the antibody via either amaleimido-caproyl group (closed circles) or a self-stabilizingmaleimido-DPR group (open circles). Tumors were allowed to reach avolume of approximately 250 mm³ before dosing at 1 mg/kg weekly forthree doses (six mice per dose group). The self-stabilizing ADC dosegroup experienced complete responses (no detectable tumor) in all sixanimals, with five animals experiencing durable regressions, while themaleimido-caproyl ADC experienced no complete responses.

DETAILED DESCRIPTION Abbreviations and Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings. When trade names are usedherein, the trade name includes the product formulation, the genericdrug, and the active pharmaceutical ingredient(s) of the trade nameproduct, unless otherwise indicated by context.

The term “electron-withdrawing group” refers to a functional group thatdraws electrons away from a reaction center. Exemplary electronwithdrawing groups include, but are not limited to, —C(═O), —CN, —NO₂,—CX₃, —X, —COOR, —CONR₂, —COR, —COX, —SO₂R, —SO₂OR, —SO₂NHR, —SO₂NR₂,—PO₃R², —P(O)(CH₃)NHR, NO, —NR₃₊, —CR═CR₂, and —C≡CR wherein X is F, Br,Cl, or I, and R is, at each occurrence, independently selected from thegroup consisting of hydrogen and C₁₋₆ alkyl. Exemplary electronwithdrawing groups can also include aryl groups (e.g., phenyl) andcertain heteroaryl groups (e.g., pyridine). The term “electronwithdrawing groups” includes aryls or heteroaryls further substituedwith electron withdrawing groups. Preferred electron withdrawing groupsare —C(═O), —CN, —NO₂, —CX₃, and —X.

The term “base” refers to a functional group that deprotonates water toproduce a hydroxide ion. Exemplary bases are amines and nitrogencontaining heterocycles. Representative bases include —N(R³)(R⁴) whereinR³ and R⁴ are independently selected from H or C₁₋₆ alkyl, preferably Hor methyl,

wherein R⁵, R⁶, R⁷ and R⁸ are, at each occurrence, independentlyselected from hydrogen or C₁₋₆ alkyl, preferably H or methyl, and e is0-4. In some aspects, the base is a nitrogenous base.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,monospecific antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments that exhibit the desired biologicalactivity. An intact antibody has primarily two regions: a variableregion and a constant region. The variable region binds to and interactswith a target antigen. The variable region includes a complementarydetermining region (CDR) that recognizes and binds to a specific bindingsite on a particular antigen. The constant region may be recognized byand interact with the immune system (see, e.g., Janeway et al., 2001,Immuno. Biology, 5th Ed., Garland Publishing, New York). An antibody canbe of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The antibody can bederived from any suitable species. In some embodiments, the antibody isof human or murine origin. An antibody can be, for example, human,humanized or chimeric.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method.

An “intact antibody” is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2, C_(H)3 and C_(H)4, as appropriate forthe antibody class. The constant domains may be native sequence constantdomains (e.g., human native sequence constant domains) or amino acidsequence variant thereof.

An “antibody fragment” comprises a portion of an intact antibody,comprising the antigen-binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments,diabodies, triabodies, tetrabodies, linear antibodies, single-chainantibody molecules, scFv, scFv-Fc, multispecific antibody fragmentsformed from antibody fragment(s), a fragment(s) produced by a Fabexpression library, or an epitope-binding fragments of any of the abovewhich immunospecifically bind to a target antigen (e.g., a cancer cellantigen, a viral antigen or a microbial antigen).

An “antigen” is an entity to which an antibody specifically binds.

The terms “specific binding” and “specifically binds” mean that theantibody or antibody derivative will bind, in a highly selective manner,with its corresponding target antigen and not with the multitude ofother antigens. Typically, the antibody or antibody derivative bindswith an affinity of at least about 1×10⁻⁷ M, and preferably 10⁻⁸ M to10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M and binds to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.

The term “inhibit” or “inhibition of” means to a reduce by a measurableamount, or to prevent entirely.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may inhibit growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can, for example, be measured by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The term “substantial” or “substantially” refers to a majority,i.e. >50% of a population, of a mixture or a sample, preferably morethan 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% of a population.

The terms “intracellularly cleaved” and “intracellular cleavage” referto a metabolic process or reaction inside a cell on a Ligand Drugconjugate (e.g., an Antibody Drug Conjugate (ADC) or the like), wherebythe covalent attachment, e.g., the linker, between the Drug moiety (D)and the Ligand unit (e.g., an antibody (Ab)) is broken, resulting in thefree Drug, or other metabolite of the conjugate dissociated from theantibody inside the cell. The cleaved moieties of the Drug-Linker-Ligandconjugate are thus intracellular metabolites.

The term “cytotoxic activity” refers to a cell-killing, a cytostatic oran anti-proliferative effect of a Drug-Linker-Ligand conjugate compoundor an intracellular metabolite of a Drug-Linker-Ligand conjugate.Cytotoxic activity may be expressed as the IC₅₀ value, which is theconcentration (molar or mass) per unit volume at which half the cellssurvive.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or inhibits the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰C, andradioactive isotopes of Lu), chemotherapeutic agents, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including synthetic analogs andderivatives thereof.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition or disorder in mammals that is typicallycharacterized by unregulated cell growth. A “tumor” comprises one ormore cancerous cells.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or proteins.

Examples of a “patient” include, but are not limited to, a human, rat,mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird andfowl. In an exemplary embodiment, the patient is a human.

The terms “treat” or “treatment,” unless otherwise indicated by context,refer to therapeutic treatment and prophylactic measures to preventrelapse, wherein the object is to inhibit or slow down (lessen) anundesired physiological change or disorder, such as the development orspread of cancer. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder.

In the context of cancer, the term “treating” includes any or all of:inhibiting growth of tumor cells, cancer cells, or of a tumor;inhibiting replication of tumor cells or cancer cells, lessening ofoverall tumor burden or decreasing the number of cancerous cells, andameliorating one or more symptoms associated with the disease.

In the context of an autoimmune disease, the term “treating” includesany or all of: inhibiting replication of cells associated with anautoimmune disease state including, but not limited to, cells thatproduce an autoimmune antibody, lessening the autoimmune-antibody burdenand ameliorating one or more symptoms of an autoimmune disease.

As used herein, the term “Detection unit” refers to refers to anymolecule which produces, or can be induced to produce, a detectablesignal. Detection units having reporter molecules that can be detectedby imaging equipment include, but are not limited to, radioactive,paramagnetic, fluorescent or radioopaque chemical entities. In someembodiments, the Detection unit will be a radioactive compound, achemiluminescent agent, a fluorescent agent, or a chromogen. In someembodiments, the Detection unit will be a fluorescent molecule such as afluorophore.

As used herein, the term “Stability unit” refers to a compound thatpromotes the stability of the conjugate, e.g., by increasing systemicretention of the Ligand when administered to a patient. A Stability unitcan also increase the water solubility of the conjugate. An exemplaryStability unit is polyethylene glycol.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound(e.g., a Drug, Drug-Linker, or a Ligand-Drug Conjugate). The compoundcan contain at least one amino group, and accordingly acid additionsalts can be formed with the amino group. Exemplary salts include, butare not limited to, sulfate, trifluoroacetate, citrate, acetate,oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counterion. The counterion may beany organic or inorganic moiety that stabilizes the charge on the parentcompound. Furthermore, a pharmaceutically acceptable salt may have morethan one charged atom in its structure. Instances where multiple chargedatoms are part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counterion.

Unless otherwise indicated, the term “alkyl” by itself or as part ofanother term refers to a substituted or unsubstituted straight chain orbranched, saturated or unsaturated hydrocarbon having the indicatednumber of carbon atoms (e.g., “—C₁-C₈ alkyl” or “—C₁-C₀₀” alkyl refer toan alkyl group having from 1 to 8 or 1 to 10 carbon atoms,respectively). When the number of carbon atoms is not indicated, thealkyl group has from 1 to 8 carbon atoms. Representative straight chain“—C₁-C₈ alkyl” groups include, but are not limited to, -methyl, -ethyl,-n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl and -n-octyl; whilebranched —C₁-C₈ alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl;unsaturated —C₂-C₈ alkyls include, but are not limited to, -vinyl,-allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,-3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl,-1-hexyl, 2-hexyl, -3-hexyl, -acetylenyl, -propynyl, -1-butynyl,-2-butynyl, -1-pentynyl, -2-pentynyl and -3-methyl-1 butynyl. In someembodiments, an alkyl group is unsubstituted. An alkyl group can besubstituted with one or more groups. In some aspects, an alkyl groupwill be saturated.

Unless otherwise indicated, “alkylene,” by itself of as part of anotherterm, refers to a substituted or unsubstituted saturated, branched orstraight chain or cyclic hydrocarbon radical of the stated number ofcarbon atoms, typically 1-10 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—), 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like. In preferred aspects, an alkylene is a branched or straightchain hydrocarbon (i.e., it is not a cyclic hydrocarbon).

Unless otherwise indicated, “aryl,” by itself or as part of anotherterm, means a substituted or unsubstituted monovalent carbocyclicaromatic hydrocarbon radical of 6-20 carbon atoms derived by the removalof one hydrogen atom from a single carbon atom of a parent aromatic ringsystem. Some aryl groups are represented in the exemplary structures as“Ar”. Typical aryl groups include, but are not limited to, radicalsderived from benzene, substituted benzene, naphthalene, anthracene,biphenyl, and the like. An exemplary aryl group is a phenyl group asfollows:

Unless otherwise indicated, an “arylene,” by itself or as part ofanother term, is an aryl group as defined above which has two covalentbonds (i.e., it is divalent) and can be in the ortho, meta, or paraconfigurations as shown in the following structures, with phenyl as theexemplary group:

Unless otherwise indicated, a “C₃-C₈ heterocycle,” by itself or as partof another term, refers to a monovalent substituted or unsubstitutedaromatic or non-aromatic monocyclic or bicyclic ring system having from3 to 8 carbon atoms (also referred to as ring members) and one to fourheteroatom ring members independently selected from N, O, P or S, andderived by removal of one hydrogen atom from a ring atom of a parentring system. One or more N, C or S atoms in the heterocycle can beoxidized. The ring that includes the heteroatom can be aromatic ornonaromatic. Unless otherwise noted, the heterocycle is attached to itspendant group at any heteroatom or carbon atom that results in a stablestructure. Representative examples of a C₃-C₈ heterocycle include, butare not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpholinyl,tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene,indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl,thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl,pyridazinyl, isothiazolyl, and isoxazolyl. A “C₃-C₈ heteroaryl,” is anaromatic C₃-C₈ heterocycle.

Unless otherwise indicated, “C₃-C₈ heterocyclo,” by itself or as part ofanother term, refers to a C₃-C₈ heterocycle group defined above whereinone of the heterocycle group's hydrogen atoms is replaced with a bond(i.e., it is divalent). A “C₃-C₈ heteroarylene,” by itself or as part ofanother term, refers to a C₃-C₈ heteroaryl group defined above whereinone of the heteroaryl group's hydrogen atoms is replaced with a bond(i.e., it is divalent).

Unless otherwise indicated, a “C₃-C₈ carbocycle,” by itself or as partof another term, is a 3-, 4-, 5-, 6-, 7- or 8-membered monovalent,substituted or unsubstituted, saturated or unsaturated non-aromaticmonocyclic or bicyclic carbocyclic ring derived by the removal of onehydrogen atom from a ring atom of a parent ring system. Representative—C₃-C₈ carbocycles include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl,1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, andcyclooctadienyl.

Unless otherwise indicated, a “C₃-C₈ carbocyclo,” by itself or as partof another term, refers to a C₃-C₈ carbocycle group defined abovewherein another of the carbocycle groups' hydrogen atoms is replacedwith a bond (i.e., it is divalent).

Unless otherwise indicated, the term “heteroalkyl,” by itself or incombination with another term, means, unless otherwise stated, a stablestraight or branched chain hydrocarbon, or combinations thereof, fullysaturated or containing from 1 to 3 degrees of unsaturation, consistingof the stated number of carbon atoms and from one to ten, preferably oneto three, heteroatoms selected from the group consisting of O, N, Si andS, and wherein the nitrogen and sulfur atoms may optionally be oxidizedand the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N and S may be placed at any interior position of theheteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. The heteroatom Si may beplaced at any position of the heteroalkyl group, including the positionat which the alkyl group is attached to the remainder of the molecule.Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃, —NH—CH₂—CH₂—NH—C(O)—CH₂—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—O—CH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. In preferred embodiments,a C₁ to C₄ heteroalkyl or heteroalkylene has 1 to 4 carbon atoms and 1or 2 heteroatoms and a C₁ to C₃ heteroalkyl or heteroalkylene has 1 to 4carbon atoms and 1 or 2 heteroatoms. In some aspects, a heteroalkyl orheteroalkylene is saturated.

Unless otherwise indicated, the term “heteroalkylene” by itself or aspart of another substituent means a divalent group derived fromheteroalkyl (as discussed above), as exemplified by —CH₂—CH₂—S—CH₂—CH₂—and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms canalso occupy either or both of the chain termini. Still further, foralkylene and heteroalkylene linking groups, no orientation of thelinking group is implied.

“Substituted alkyl” and “substituted aryl” mean alkyl and aryl,respectively, in which one or more hydrogen atoms are each independentlyreplaced with a substituent. Typical substituents include, but are notlimited to, —X, —R, —O—, —OR, —SR, —S—, —NR₂, —NR₃, ═NR, —CX₃, —CN,—OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, —NRC(═O)R, —C(═O)R,—C(═O)NR₂, —SO₃—, —SO₃H, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR, —S(═O)R,—OP(═O)(OR)₂, —P(═O)(OR)₂, —PO⁻ ₃, —PO₃H₂, —AsO₂H₂, —C(═O)R, —C(═O)X,—C(═S)R, —CO₂R, —CO₂, —C(═S)OR, —C(═O)SR, —C(═S)SR, —C(═O)NR₂,—C(═S)NR₂, or —C(═NR)NR₂, where each X is independently a halogen: —F,—Cl, —Br, or —I; and each R is independently —H, —C₁-C₂₀ alkyl, —C₆-C₂₀aryl, —C₃-C₁₄ heterocycle, a protecting group or a prodrug moiety.Alkylene, carbocycle, carbocyclo, arylene, heteroalkyl, heteroalkylene,heterocycle, heterocyclo, heteroaryl, and heteroarylene groups asdescribed above may also be similarly substituted.

RG is a reactive group that contains a reactive site (RS) that iscapable of forming a bond with either the components of the Linker unit(i.e., A, W, Y) or the Drug unit D. RS is the reactive site within aReactive Group (RG). Reactive groups include sulfhydryl groups to formdisulfide bonds or thioether bonds, aldehyde, ketone, or hydrazinegroups to form hydrazone bonds, carboxylic or amino groups to formpeptide bonds, carboxylic or hydroxy groups to form ester bonds,sulfonic acids to form sulfonamide bonds, alcohols to form carbamatebonds, and amines to form sulfonamide bonds or carbamate bonds. Thefollowing table is illustrative of Reactive Groups, Reactive Sites, andexemplary functional groups that can form after reaction of the reactivesite. The table is not limiting. One of skill in the art will appreciatethat the noted R′ and R″ portions in the table are effectively anyorganic moiety (e.g., an alkyl group, aryl group, heteroaryl group, orsubstituted alkyl, aryl, or heteroaryl, group) which is compatible withthe bond formation provided in converting RG to one of the ExemplaryFunctional Groups. It will also be appreciated that, as applied to theembodiments of the present invention, R′ may represent one or morecomponents of the self-stabilizing linker or optional secondary linker,as the case may be, and R″ may represent one or more components of theoptional secondary linker, drug unit, stabilizing unit, or detectionunit, as the case may be.

Exemplary RG RS Functional Groups 1) R′—SH —S— R′—S—R″ R′—S—S—R″ 2)R′—C(═O)OH —C(═O)— R′—C(═O)NH—R″ 3) R′—C(═O)ONHS —C(═O)— R′—C(═O)NH—R″4) R′S(═O)₂—OH —S(═O)₂— R′S(═O)₂NH—R″ 5) R′—CH₂—X (X is Br, I, Cl) —CH₂—R′—CH₂—S—R″ 6) R′—NH₂ —N— R′—NHC(═O)R″It will be understood that, once reacted, the reactive site RS can forma new bond with components of the Linker unit or the Drug unit, as thecase may be. The reactive site, RS, once linked to the remainder of theLinker unit has typically lost its reactivity.

The term “dilactam” as used herein refers to a cyclic amide that formsfrom a macro-cyclicization reaction with a thio-substituted succinimideand base present on the self-stabilizing linker assembly.

General

Hydrolysis of a maleimide (or thio-substituted succinimide) represents anucleophilic addition reaction in which water, acting as thenucleophile, attacks one of the electrophilic carbonyl carbon atoms ofthe maleimide ring (or succinimide ring). The rate of this reaction isinfluenced by electrophilicity of the carbonyls, which can vary with thesubstitution of electron-donating or electron-withdrawing groups presenton the nitrogen of the imide group. The rate of the hydrolysis reactionis also influenced by the pH of an aqueous solvent, which effectivelyincreases the nucleophilicity of water with increasing pH. It has beendiscovered by the present inventors that the placement of a basic groupon an N-substituted maleimide also increases the rate of the hydrolysis.By careful engineering of an N-substituent group on the maleimide, thecombination of its electron withdrawing influence on the maleimide ring(thus increasing its electrophilicity) and localized basicity(increasing the effective nucleophilicity of nearby water) can be usedto tune the rate of hydrolysis of either the parent maleimide or itsthio-substituted succinimide derivative. The present invention provides,inter alia, N-substituted maleimides with hydrolysis rates that fallwithin a useful range wherein their reaction with thiols occurs morequickly than their hydrolysis to the maleic acid derivative, but whichyield thio-substituted succinimides with hydrolysis rates that aresufficiently rapid to achieve complete hydrolysis under gentleconditions that are very suitable for the manufacture of protein-basedbioconjugates.

The present invention is based, in part, on the discovery that a basicfunctional group proximal to a maleimide will catalyze the hydrolysis ofa thio-substituted succinimide which is formed upon conjugation of themaleimide and a protein thiol leading to a stable bioconjugate. Byfurther combining a proximal basic group with an electron withdrawinggroup, the rate of thio-substituted succinimide ring hydrolysis can betuned to a desirable level. Design parameters that affect the rate ofhydrolysis include the pKa of the basic group, the strength of theelectron withdrawing group when present, and the proximity of bothgroups to the maleimide carbonyl carbons. Design parameters that affectthe percentage hydrolysis include the nature and proximity of the baseto the maleimide carbonyl carbons.

Conceptually, without limiting the invention, a Linker unit comprising aself-stabilizing linker assembly is referred to herein as aSelf-Stabilizing Linker or Self-Stabilizing Linker unit. TheSelf-Stabilizing Linker prior to conjugation with the Ligand unitcomprises a maleimide group. The Self-Stabilizing Linker isself-stabilizing by virtue of the proximity of the maleimide group to abase within the Linker unit which catalyzes the hydrolysis of its ownthio-substituted succinimide after conjugation to the Ligand unit. Thisis represented schematically below:

It will be understood that the term Self-Stabilizing Linker refers tothe Linker unit both prior to and post stabilization.

In view of the above, the present invention provides in one group ofembodiments, a Ligand-Functional Agent Conjugate comprising a Ligandunit and at least one Functional Agent selected from a Drug unit, aDetection Unit, or a Stabilizing Unit, wherein the Ligand unit and eachof the Functional Agent(s) are joined by a self-stabilizing linkerassembly comprising a succinimide ring or a hydrolyzed succinimide ringdirectly conjugated to the Ligand unit via a thioether linkage; and abase and an electron withdrawing group (conjugated to the Ligand unitvia the succinimide) operably linked to stabilize the conjugate inplasma relative to a ligand drug conjugate lacking the self-stabilizinglinker assembly (i.e. by increasing the rate of succinimide ringhydrolysis). In some aspects, the electron withdrawing group ispositioned to increase the electrophilicity of the succinimide renderingit more reactive with water and the base is positioned to assist thehydrolysis of the succinimide ring (e.g., by an intramolecular basecatalysis mechanism). In some aspects, in place of the succinimide ringis a dilactam formed when the base reacts with the succinimide ring. Inanother group of embodiments, Functional Agent-Linker units are providedwherein the Linker portion comprises a self-stabilizing linker assembly.In another group of embodiments, Ligand-Linker conjugates are provided,wherein the Linker portion comprises a self-stabilizing linker assembly.In some embodiments, the Linker portion further comprises an optionalsecondary linker assembly (L^(O)).

In some aspects, the Ligand-Functional Agent Conjugate is a Ligand-DrugConjugate. Accordingly, the present invention provides in one group ofembodiments, a Ligand-Drug Conjugate comprising a Ligand unit and atleast one Drug unit, wherein the Ligand unit and each of the Drugunit(s) are joined by a self-stabilizing linker assembly comprising asuccinimide ring or a hydrolyzed succinimide ring directly conjugated tothe Ligand unit via a thioether linkage; and a base and an electronwithdrawing group (conjugated to the Ligand unit via the succinimidering) operably linked to stabilize the conjugate in plasma relative to aligand drug conjugate lacking the self-stabilizing linker assembly (i.e.by increasing the rate of succinimide ring hydrolysis). In some aspects,the electron withdrawing group is positioned to increase theelectrophilicity of the succinimide rendering it more reactive withwater and the base is positioned to assist the hydrolysis of thesuccinimide ring (e.g., by an intramolecular base catalysis mechanism).In some aspects, in place of the succinimide ring is a dilactam formedwhen the base reacts with the succinimide ring. In another group ofembodiments, Drug-Linker units are provided wherein the Linker portioncomprises a self-stabilizing linker assembly. In another group ofembodiments, Ligand-Linker conjugates are provided, wherein the Linkerportion comprises a self-stabilizing linker assembly. In someembodiments, the Linker portion further comprises an optional secondarylinker assembly (L^(O)). In some embodiments, the secondary linkerassembly is a releasable linker assembly (L^(R)) which comprises aCleavable unit and optionally one or more of a Stretcher and a Spacerunit. In some other embodiments, the secondary linker assembly is anon-releasable linker assembly (L^(N)) which comprises one or more of aStretcher unit and a Spacer unit. In still other embodiments, theinvention provides methods of treating cancer, immune disease,infectious diseases and other diseases and disorders using a Ligand-DrugConjugate comprising a self-stabilizing linker assembly.

The Linker unit of the Ligand-Functional Agent Conjugate (or Ligand-DrugConjugate) can further comprise, in addition to a self-stabilizinglinker assembly, an optional secondary linker assembly (L^(O)) whichjoins each Functional Agent (or Drug unit) to the self-stabilizinglinker assembly. The secondary linker assembly can be a releasablelinker assembly or a non-releasable linker assembly.

The term Linker unit can be used herein to refer to the linker portionof the Ligand-Functional Agent Conjugate (or Ligand-Drug Conjugate)comprising the self-stabilizing linker assembly and optional secondarylinker assembly.

The Self-Stabilizing Linker Assembly

The Self-Stabilizing Linkers are designed such that the rate of thepost-conjugation hydrolysis of the succinimide ring will be controllableand fall within a desired range. The limits of this range are typicallydictated by issues which arise in the manufacture of ligand-drugconjugates. On the one hand, hydrolysis which is too slow would requireunacceptable delays in the manufacturing process or aggressiveconditions of pH and temperature which may induce damage to the proteinbackbone. Conversely, a maleimide which is too reactive with water maybe hydrolyzed to the corresponding maleic acid derivative before it canreact with available protein thiols (see undesired pathway):

Such maleic acid derivatives are not reactive with thiols, and thus thisreaction pathway does not result in a bioconjugate. Therefore,maleimides which undergo hydrolysis faster than thiol addition underapplicable conditions are not useful reagents. In general, structuralfeatures which increase the hydrolysis rate of a thio-substitutedsuccinimide will also increase the hydrolysis rate of the parentmaleimide.

In designing the Self-Stabilizing Linkers of the present invention, itwill be understood that the pKa of the basic group, the strength of theelectron withdrawing group(s), and the proximity of both groups to themaleimide are inter-related variables and will affect the hydrolysisrate of both the maleimide and corresponding thio-substitutedsuccinimide product. Accordingly, positioning of the electronwithdrawing group and base will be dependent upon the pKa of the baseand the strength of the electron withdrawing group(s). The skilledartisan will understand that for particularly strong electronwithdrawing groups such as fluoro, trifluoromethyl, and nitro, the groupcan be further from the maleimide. In some embodiments, the hydrolysisreaction may compete with a macro-cyclization reaction such that theresultant conjugates comprise a heterogenous mixture of hydrolyzedthio-substituted succinimide conjugates and cyclized thio-substituteddilactam conjugates. In preferred embodiments, a dilactam will not beformed.

Selected Embodiments of the Invention

In some embodiments, the Ligand-Functional Agent Conjugate isrepresented by Formula I:

or a salt thereof (e.g., pharmaceutically acceptable salt thereof);whereinL is a Ligand unit;D′ is a Drug unit, a Detection unit, or a Stabilizing unit;L^(O) is the optional secondary linker assembly; andL^(SS) is the self-stabilizing linker assembly, wherein

-   -   M¹ is a succinimide ring or a hydrolyzed succinimide or together        with BU forms a dilactam;    -   BU is a Basic unit;    -   HE is a hydrolysis enhancer comprising an electron withdrawing        group;    -   the circle represents a scaffold that can be C₁₋₈ alkylene, C₁₋₈        heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and        optionally comprises a reactive site suitable for attachment to        the optional secondary linker assembly or D′;    -   the subscripts m, q and r are each 0 or 1, and the sum of m+q+r        is 0, 1 or 2 provided that if m+q+r is 0, the scaffold is a        C₆₋₁₀ arylene or C₄₋₁₀ heteroarylene;    -   the subscript a and b are each 0 or 1, and the sum of a+b is 1;        and    -   the subscript p ranges from 1 to 20.

In some aspects, when r is 1, HE does not comprise a carbonyl group,(i.e., C(═O))

In some aspects, r is zero.

In some aspects m+q+r is 0. In such aspects, the C₆₋₁₀ arylene or C₄₋₁₀heteroarylene act as the electron withdrawing group. Exemplary aryls andheteroaryls include phenyl and pyridinyl.

In some aspects m+q+r is 1 or 2.

In some aspects, the Conjugate is represented by Formula I or a saltthereof wherein a is 1 and r is zero.

In some aspects, the Conjugate is represented by Formula I or a saltthereof wherein L^(O) is present and is a releasable linker assembly,the circle represents a scaffold that is C₁₋₈ alkylene or C₁₋₈heteroalkylene (preferably C₁₋₄ alkylene or C₁₋₄ heteroalkylene), a is1, r is zero, and the sum of m+q is 1. In some such aspects, thescaffold is C₁₋₃ alkylene or C₁₋₃ heteroalkylene. In some such aspects,the alkylene is straight chain or branched.

In some aspects, the Conjugate is represented by Formula I or a saltthereof wherein L^(O) is present and is a releasable linker assembly,the circle represents a scaffold that is C₁₋₈ alkylene or C₁₋₈heteroalkylene (preferably C₁₋₄ alkylene or C₁₋₄ heteroalkylene), a is1, and m and r are zero. In some such aspects, the scaffold is C₁₋₃alkylene or C₁₋₃ heteroalkylene. In some such aspects, the alkylene isstraight chain or branched.

In some aspects, the Conjugate is represented by Formula I or a saltthereof wherein L^(O) is present and is a releasable linker assembly,the circle represents a scaffold that is C₁, C₂, C₃ or C₄ straight orbranched chain alkylene, a is 1, r is zero, and the sum of m+q is 1.

In some aspects, the Conjugate is represented by Formula I or a saltthereof wherein L^(O) is present and is a releasable linker assembly,the circle represents a scaffold that is C₁, C₂, C₃ or C₄ straight orbranched chain alkylene, a is 1, and m and r are zero.

In some aspects, there are no less than 2 and no more than 6 interveningatoms between the base of the Basic unit and the nitrogen atom of thesuccinimide (hydrolyzed or non-hydrolyzed) or dilactam and there are nomore than 5 atoms, no more than 4 atoms, no more than 3 atoms, or nomore than 2 intervening atoms between the electron withdrawing group andthe nitrogen atom of the succinimide ring (hydrolyzed or non-hydrolyzed)or dilactam.

In each of these embodiments, the alkylene or heteroalkylene chain canbe straight or branched. In some aspects, the alkylene or heteroalkylenechain will be a straight chain. In other aspects, it will be branched.

In each of these embodiments, p can range from 1 to 20, preferably 1 to12, even more 1 to 10 or 1 to 8.

In each of these embodiments, M¹ is preferably a succinimide ring (i.e.,non-hydrolyzed) or a hydrolyzed succinimide ring (also referred toherein as hydrolyzed succinimide).

In each of these embodiments, D′ can be a Drug unit, D, and theLigand-Functional Agent Conjugate can be a Ligand-Drug conjugate.

In some aspects wherein the scaffold itself is directly linked to theoptional secondary linker assembly or D′, (for example, in selectembodiments when q is zero or when q is zero and r is zero), thescaffold will comprise a reactive site suitable for attachment to theoptional secondary linker assembly or D′.

In some embodiments, the self-stabilizing linker assembly (L^(SS)) isrepresented by Formula II:

or a salt thereof (e.g., pharmaceutically acceptable salt) wherein thewavy lines indicates points for attachment of the optional secondarylinker assembly to D′ or D, and wherein // indicates the point ofattachment to a Ligand Unit. In the self-stabilizing linker assemblyabove, M¹ represents a succinimide ring or a hydrolyzed succinimide ringor a dilactam formed when the base reacts with the succinimide ring, BUis a Basic unit, HE is a hydrolysis enhancer comprising an electronwithdrawing group, and the circle represents a scaffold that can be C₁₋₈alkylene, C₁₋₈ heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene,and optionally comprises a reactive site suitable for attachment to theoptional secondary linker assembly, D′, or D; and the subscripts m, qand r are each 0 or 1, and the sum of m+q+r is 0, 1 or 2 provided thatif m+q+r is 0, the scaffold is a C₆₋₁₀ arylene or C₄₋₁₀ heteroarylene.

In some aspects, when r is 1, HE does not comprise a carbonyl group,(i.e., C(═O))

In some aspects, the self-stabilizing linker assembly is represented byFormula II wherein r is zero.

In some aspects m+q+r is 0. In such aspects, the C₆₋₁₀ arylene or C₄₋₁₀heteroarylene act as the electron withdrawing group. Exemplary aryls andheteroaryls include phenyl and pyridinyl.

In some aspects m+q+r is 1 or 2.

In some aspects, the self-stabilizing linker assembly is represented byFormula II or a salt thereof wherein the circle represents a scaffoldthat is C₁₋₈ alkylene or C₁₋₈ heteroalkylene (preferably C₁₋₄ alkyleneor heteroalkylene), r is zero, and the sum of m+q is 1. In some suchaspects, the scaffold is C₁₋₃ alkylene or C₁₋₃ heteroalkylene. In somesuch aspects, the alkylene is a straight chain or branched alkylene.

In some aspects, the self-stabilizing linker assembly is represented byFormula II or a salt thereof wherein, the circle represents a scaffoldthat is C₁₋₈ alkylene or C₁₋₈ heteroalkylene (preferably C₁₋₄ alkyleneor heteroalkylene) and m and r are zero. In some such aspects, thescaffold is C₁₋₃ alkylene or C₁₋₃ heteroalkylene. In some such aspects,the alkylene is a straight chain or branched alkylene.

In some aspects, the self-stabilizing linker assembly is represented byFormula II or a salt thereof wherein the circle represents a scaffoldthat is C₁, C₂, C₃, or C₄ straight or branched chain alkylene, r iszero, and the sum of m+q is 1.

In some aspects, the self-stabilizing linker assembly is represented byFormula II or a salt thereof wherein the circle represents a scaffoldthat is C₁, C₂, C₃, or C₄ straight or branched chain alkylene, and m andr are zero.

In some aspects, there are no less than 2 and no more than 6 interveningatoms between the base of the Basic unit and the nitrogen atom of thesuccinimide (hydrolyzed or non-hydrolyzed) or dilactam and there are nomore than 5 atoms, no more than 4 atoms, no more than 3 atoms, or nomore than 2 intervening atoms between the electron withdrawing group andthe nitrogen atom of the succinimide ring (hydrolyzed or non-hydrolyzed)or dilactam.

In each of these embodiments, the alkylene or heteroalkylene chain willbe a straight or branched chain. In some aspects, the alkylene orheteroalkylene chain will be a straight chain. In other aspects, it willbe a branched chain.

In each of these embodiments, M¹ is preferably a succinimide ring or ahydrolyzed succinimide ring.

In each of these embodiments, D′ is preferably D, a Drug unit.

Returning to the embodiments of the invention wherein theLigand-Functional Agent Conjugate has the Formula (I):

or a salt thereof, wherein each of the scaffold, L, M¹, HE, BU, L^(O),D′, and the subscripts p, a, b, m, q and r have the meanings providedabove, selected embodiments include those wherein:1) m is 1, and q and r are 0;2) q is 1, and m and r are 0;3) r is 1, and m and q are 0;4) m is 1, q and r are 0, and a is 1;5) q is 1, m and r are 0, and a is 1;6) r is 1, m and q are 0, and a is 1;7) m is 1, q and r are 0, and D′ is a Drug unit, D;8) q is 1, m and r are 0, and D′ is a Drug unit, D;9) r is 1, m and q are 0, and D′ is a Drug unit, D;10) m is 1, q and r are 0, a is 1, and D′ is a Drug unit, D;11) q is 1, m and r are 0, a is 1, and D′ is a Drug unit, D; or12) r is 1, m and q are 0, a is 1, and D′ is a Drug unit, D.

In other selected embodiments, including each of the selectedembodiments of 1), 2) 3), 4), 5), 6), 7), 8), 9), 10), 11), and 12)above, the Basic unit (BU) comprises a primary, a secondary amine, or atertiary amine. In still other selected embodiments, including each ofthe selected embodiments of 1), 2) 3), 4), 5), 6), 7), 8), 9), 10), 11),and 12) above, the Basic unit is selected from the group consisting of—(C(R⁹)(R¹⁰))_(x)NH₂, —(C(R⁹)(R¹⁰))_(x)NHR^(a), and—(C(R⁹)(R¹⁰))_(x)NR^(a) ₂, wherein x is an integer of from 0-4 (or from1 to 4) and each R^(a) is independently selected from the groupconsisting of C₁₋₆ alkyl and C₁₋₆ haloalkyl, or two R^(a) groups arecombined with the nitrogen to which they are attached to form anazetidinyl, pyrrolidinyl or piperidinyl group, provided that if x iszero there are no less than 2 intervening atoms between the base of theBasic unit and the nitrogen atom of the succinimide (hydrolyzed ornon-hydrolyzed) or dilactam, and each R⁹ and R¹⁰ are independentlyselected from H or C₁₋₃ alkyl. In still other selected embodiments,including each of the selected embodiments of 1), 2) 3), 4), 5), 6), 7),8), 9), 10), 11), and 12) above, the Basic unit is selected from thegroup consisting of —(CH₂)_(x)NH₂, —(CH₂)_(x)NHR^(a), and—(CH₂)_(x)NR^(a) ₂, wherein x is an integer of from 0 to 6 (preferably 0to 4, or 1 to 4) provided that if x is zero there are no less than 2intervening atoms between the base of the Basic unit and the nitrogenatom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, andeach R^(a) is independently selected from the group consisting of C₁₋₆alkyl and C₁₋₆ haloalkyl, or two R^(a) groups are combined with thenitrogen to which they are attached to form an azetidinyl, pyrrolidinylor piperidinyl group. In yet other selected embodiments, x is an integerof from 1 to 4. In even other selected embodiments, including each ofthe selected embodiments of 1), 2) 3), 4), 5), 6), 7), 8), 9), 10), 11),and 12) above, the Basic unit is —NH₂, —CH₂NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂NH₂, or —CH₂CH₂CH₂CH₂ NH₂ provided that if the Basic unit is —NH₂, thereare no less than 2 intervening atoms between the base and the nitrogenatom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam.

In still other selected embodiments, including the selected embodimentsof 2), 5), 8), and 11) above and including the embodiments of thepreceding paragraph, HE comprises a carbonyl, sulfonyl or phosphorylmoiety.

In yet other selected embodiments, including each of the selectedembodiments above (e.g., each of the selected embodiments of 1), 2) 3),4), 5), 6), 7), 8), 9), 10), 11), and 12) above and including theembodiments of the preceding paragraphs), there are no less than 2 andno more than 6 intervening atoms between the base of the Basic unit andthe nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) ordilactam and there are no more than 5 atoms, no more than 4 atoms, nomore than 3 atoms, or no more than 2 intervening atoms between theelectron withdrawing group and the nitrogen atom of the succinimide ring(hydrolyzed or non-hydrolyzed) or dilactam.

In yet other selected embodiments, including each of the selectedembodiments above (e.g., each of the selected embodiments of 1), 2) 3),4), 5), 6), 7), 8), 9), 10), 11), and 12) above and including theembodiments of the preceding paragraphs, M¹ is a succinimide ring orhydrolyzed succinimide.

In yet other selected embodiments, including each of the selectedembodiments above (e.g., each of the selected embodiments of 1), 2) 3),4), 5), 6), 7), 8), 9), 10), 11), and 12) above and including theembodiments of the preceding paragraphs, the circle represents ascaffold that is C₁₋₈ alkylene or C₁₋₈ heteroalkylene (preferably C₁₋₄alkylene or C₁₋₄ heteroalkylene). In some such aspects the alkylene is astraight or branched chain alkylene.

In yet other selected embodiments, the Ligand-Functional AgentConjugates have the formula:

or a pharmaceutically acceptable salt thereof, wherein each of thescaffold, L, M¹, HE, BU, L^(O), D′, and the subscript p has the meaningprovided above, selected embodiments include those wherein:1) the Basic unit (BU) comprises a primary, a secondary amine, or atertiary amine, and D′ is preferably a Drug unit D.2) the Basic unit is selected from the group consisting of—(C(R⁹)(R¹⁰))_(x)NH₂, —(C(R⁹)(R¹⁰))_(x)NHR^(a), and—(C(R⁹)(R¹⁰))_(x)NR^(a) ₂, wherein x is an integer of from 0-4 (or 1-4)and each R^(a) is independently selected from the group consisting ofC₁₋₆ alkyl and C₁₋₆ haloalkyl, or two R^(a) groups are combined with thenitrogen to which they are attached to form an azetidinyl, pyrrolidinylor piperidinyl group, provided that if x is zero, there are no less than2 intervening atoms between the base of the Basic unit and the nitrogenatom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, andR⁹ and R¹⁰ are independently selected from H or C₁₋₃ alkyl, and D′ ispreferably a Drug unit D.3) the Basic unit is selected from the group consisting of—(CH₂)_(x)NH₂, —(CH₂)_(x)NHR^(a), and —(CH₂)_(x)NR^(a) ₂, wherein x isan integer of from 0 to 6 (preferably 0 to 4 or 1 to 4) provided that ifx is zero, there are no less than 2 intervening atoms between the baseof the Basic unit and the nitrogen atom of the succinimide (hydrolyzedor non-hydrolyzed) or dilactam, and each R^(a) is independently selectedfrom the group consisting of C₁₋₆ alkyl and C₁₋₆ haloalkyl, or two R^(a)groups are combined with the nitrogen to which they are attached to forman azetidinyl, pyrrolidinyl or piperidinyl group, and D′ is preferably aDrug unit D. In yet other selected embodiments, X is an integer of from1 to 4.4) the Basic unit is —NH₂, —CH₂NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂ NH₂, or—CH₂CH₂CH₂CH₂ NH₂ provided that if the Basic unit is —NH₂, there are noless than 2 intervening atoms between the base and the nitrogen atom ofthe succinimide (hydrolyzed or non-hydrolyzed) or dilactam; and D′ ispreferably a Drug unit D.

In still other selected embodiments, including each of the selectedembodiments above, HE comprises a carbonyl, sulfonyl or phosphorylmoiety, and D′ is preferably a Drug unit D.

In yet other selected embodiments, including each of the selectedembodiments above, there are no less than 2 and no more than 6intervening atoms between the base of the Basic unit and the nitrogenatom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam andthere are no more than 5 atoms, no more than 4 atoms, no more than 3atoms, or no more than 2 intervening atoms between the electronwithdrawing group and the nitrogen atom of the succinimide ring(hydrolyzed or non-hydrolyzed) or dilactam, and D′ is preferably a Drugunit (D).

In yet other selected embodiments, including each of the selectedembodiments above, M¹ is a succinimide ring or hydrolyzed succinimide,and D′ is preferably a Drug unit (D).

In yet other selected embodiments, including each of the selectedembodiments above, the circle represents a scaffold that is C₁₋₈alkylene or C₁₋₈ heteroalkylene (preferably C₁₋₄ alkylene or C₁₋₄heteroalkylene), and D′ is preferably a Drug unit (D). In some suchaspects, the alkylene is a straight chain or branched chain alkylene.

In still other selected embodiments, including each of the selectedembodiments above, HE is a carbonyl, and D′ is preferably a Drug unit(D).

In still other selected embodiments, including each of the selectedembodiments above, HE is a carbonyl and the circle represents a scaffoldthat is a straight chain C₁₋₈ alkylene or C₁₋₈ heteroalkylene(preferably C₁₋₄ alkylene or C₁₋₄ heteroalkylene), and D′ is preferablya Drug unit (D).

In still other selected embodiments, including each of the selectedembodiments above, HE is a carbonyl and the circle represents a scaffoldthat is a branched chain C₁₋₈ alkylene or C₁₋₈ heteroalkylene(preferably C₁₋₄ alkylene or C₁₋₄ heteroalkylene), and D′ is preferablya Drug unit (D).

In yet other selected embodiments, Ligand-Drug Conjugates have theformula:

or a pharmaceutically acceptable salt thereof, wherein the Ligandportion is an antibody (Ab), the subscript p ranges from 1 to 20(preferably 1 to 12), and M¹, BU, L^(O) are as described in any of theembodiments provided herein, and D is a Drug unit. For example, in someaspects, L^(O) is a releasable linker assembly, and BU is —(CH₂)_(x)NH₂,—(CH₂)_(x)NHR^(a), and —(CH₂)_(x)NR^(a) ₂, wherein x is an integer offrom 1-4 and each R^(a) is independently selected from the groupconsisting of C₁₋₆ alkyl and C₁₋₆ haloalkyl, or two R^(a) groups arecombined with the nitrogen to which they are attached to form anazetidinyl, pyrrolidinyl or piperidinyl group. In yet other aspects,L^(O) is a releasable linker assembly, and BU is —CH₂NH₂, —CH₂CH₂NH₂,—CH₂CH₂CH₂ NH₂, or —CH₂CH₂CH₂CH₂ NH₂. In some aspects, the Ab can bereplaced by a non-antibody protein.

In yet other selected embodiments, the Ligand-Drug Conjugates have theformula:

or a pharmaceutically acceptable salt thereof wherein the Ligand portionis an antibody (Ab) and the subscript p ranges from 1 to 20 (preferably1 to 12) and M¹, BU, and L^(O) are as described in any of theembodiments provided herein, and D is a Drug unit. For example, in someaspects, L^(O) is a releasable linker assembly, and BU is —(CH₂)_(x)NH₂,—(CH₂)_(x)NHR^(a), and —(CH₂)_(x)NR^(a) ₂, wherein x is an integer offrom 1-4 and each R^(a) is independently selected from the groupconsisting of C₁₋₆ alkyl and C₁₋₆ haloalkyl, or two R^(a) groups arecombined with the nitrogen to which they are attached to form anazetidinyl, pyrrolidinyl or piperidinyl group. In yet other aspects,L^(O) is a releasable linker assembly, and BU is —CH₂NH₂, —CH₂CH₂NH₂,—CH₂CH₂CH₂ NH₂, or —CH₂CH₂CH₂CH₂ NH₂. In some aspects, the Ab can bereplaced by a non-antibody protein.

In yet other selected embodiments, the Ligand-Drug Conjugates have theformula:

or a pharmaceutically acceptable salt thereof wherein the Ligand portionis an antibody (Ab), the subscript p ranges from 1 to 20 (preferably 1to 12), and M¹, BU, andL^(O) are as described in any of the embodimentsprovided herein and D is a Drug unit. For example, in some aspects,L^(O) is a releasable linker assembly, and BU is —(CH₂)_(x)NH₂,—(CH₂)_(x)NHR^(a), and —(CH₂)_(x)NR^(a) ₂, wherein x is an integer offrom 0-4 and each R^(a) is independently selected from the groupconsisting of C₁₋₆ alkyl and C₁₋₆ haloalkyl, or two R^(a) groups arecombined with the nitrogen to which they are attached to form anazetidinyl, pyrrolidinyl or piperidinyl group. In yet other aspects,L^(O) is a releasable linker assembly, and BU is —NH₂, —CH₂NH₂,—CH₂CH₂NH₂, —CH₂CH₂CH₂ NH₂, or —CH₂CH₂CH₂CH₂ NH₂. In some aspects, theAb can be replaced by a non-antibody protein.

Having described a variety of Ligand-Functional Agent Conjugates andLigand-Drug Conjugates provided by the present disclosure, one of skillin the art will appreciate that component assemblies are also useful.Accordingly, the present invention provides Functional Agent-LinkerConjugates (e.g., Drug-Linker Conjugates), Linkers, and Ligand-Linkerassemblies.

Functional Agent-Linker Conjugates

In another embodiment, the present invention provides FunctionalAgent-Linker Conjugates (e.g., Drug-Linker Conjugates) having theformula:

or a salt thereof (e.g., pharmaceutically acceptable salt) wherein,D′ is a Drug unit, a Detection unit, or a Stabilizing unit;L^(O) is an optional secondary linker assembly; andL^(SS) is the self-stabilizing linker assembly, wherein

-   -   BU is a Basic unit;    -   HE is a hydrolysis enhancer comprising an electron withdrawing        group;    -   the circle represents a scaffold that can be C₁₋₈ alkylene, C₁₋₈        heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and        optionally comprises a reactive site suitable for attachment to        the optional secondary linker assembly or D′;    -   the subscripts m, q and r are each 0 or 1, and the sum of m+q+r        is 0, 1 or 2 provided that if m+q+r is 0, the scaffold is a        C₆₋₁₀ arylene or C₄₋₁₀ heteroarylene, and    -   the subscript a and b are each 0 or 1, and the sum of a+b is 1.

In certain selected embodiments, the Functional Agent-Linker Conjugateis represented by the formula:

or a salt thereof, while in other selected embodiments, the Drug-LinkerConjugate is represented by the formulae:

or a salt thereof, wherein the circle, HE, BU, L^(O) and D′ have themeanings provided above for Formula I and D is a Drug unit.Additionally, each of the specifically recited selected embodiments forthe circle, HE, BU, L^(O) and D′ (for Formula I or any of the conjugatesprovided herein) are equally applicable to these Drug-Linker Conjugates.In preferred aspects D′ is a Drug unit, D.

Linkers

Also provided herein are Linkers having the formula:

or a salt thereof (e.g., pharmaceutically acceptable salt), whereinRG is a reactive group (comprising a reactive site) at the terminus ofL^(O), suitable for attaching a Drug unit;L^(O) is an optional secondary linker assembly that is present; andL^(SS) is the self-stabilizing linker assembly, wherein

-   -   BU is a Basic unit;    -   HE is a hydrolysis enhancer comprising an electron withdrawing        group;    -   the circle represents a scaffold that can be C₁₋₈ alkylene, C₁₋₈        heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and        optionally comprises a reactive site suitable for attachment to        the optional secondary linker assembly or Drug unit;    -   the subscripts m, q and r are each 0 or 1, and the sum of m+q+r        is 0, 1 or 2 provided that if m+q+r is 0, the scaffold is a        C₆₋₁₀ arylene or C₄₋₁₀ heteroarylene; and    -   the subscript a and b are each 0 or 1, and the sum of a+b is 1.

In some aspects wherein the Linker is attached to a Detection unit or aStabilizing unit, RG is a reactive group that contains a reactive sitethat is capable of forming a bond with a Detection unit or a Stabilizingunit instead of a Drug unit.

In certain selected embodiments, the Linker is represented by theformula:

or a salt thereof, while in other selected embodiments, the Linker isrepresented by the formulae:

or a salt thereof (e.g., pharmaceutically acceptable salt) wherein thecircle, HE, BU, L^(O) and RG have the meanings provided above.Additionally, each of the specifically recited selected embodiments forBU, L^(O) and RG (for any of the conjugates provided herein) are equallyapplicable to these Linkers.

Ligand-Linker Conjugates

Also provided herein are Ligand-Linker Conjugates, having the formula:

or a salt thereof (e.g., pharmaceutically acceptable salt) wherein

-   L is a Ligand unit;-   the subscript p ranges from 1 to 20;-   RG is a reactive group (comprising a reactive site) at the terminus    ofL^(O) which is suitable for attaching a Drug unit;-   L^(O) is an optional secondary linker assembly that is present; and-   L^(SS) is a self-stabilizing linker assembly, wherein    -   M¹ is a succinimide ring or hydrolyzed succinimide;    -   BU is a Basic unit;    -   HE is a hydrolysis enhancer comprising an electron withdrawing        group;    -   the circle represents a scaffold that can be C₁₋₈ alkylene, C₁₋₈        heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and        optionally comprises a reactive site suitable for attachment to        the optional secondary linker assembly or Drug unit;    -   the subscripts m, q and r are each 0 or 1, and the sum of m+q+r        is 0, 1 or 2 provided that if m+q+r is 0, the scaffold is a        C₆₋₁₀ arylene or C₄₋₁₀ heteroarylene; and    -   the subscript a and b are each 0 or 1, and the sum of a+b is 1.

In some aspects wherein the Ligand-Linker Conjugate is attached to aDetection unit or a Stabilizing unit instead of a Drug unit, RG is areactive group that contains a reactive site that is capable of forminga bond with a Detection unit or a Stabilizing unit instead of a Drugunit.

In certain selected embodiments, the Ligand-Linker Conjugate isrepresented by the formula:

or a salt thereof while in other selected embodiments, the Ligand-LinkerConjugate is represented by the formula:

or a salt thereof (e.g., pharmaceutically acceptable salt), wherein L isan antibody (Ab), and the circle, HE, M¹, BU, L^(O) and RG have themeanings provided above. Additionally, each of the specifically recitedselected embodiments for Ab, M¹, BU, L^(O) and RG (for any of theconjugates provided herein) are equally applicable to theseLigand-Linker conjugates.

In some embodiments of the invention, the self-stabilizing linkerassembly instead of being represented by the structure for L^(SS) isrepresented by L^(TT) and has Formula (III):

or a pharmaceutically acceptable salt thereof, wherein the wavy lineindicates points of attachment of the optional secondary linker assemblyor Drug unit and wherein // indicates the point of attachment to aLigand unit;wherein M¹ is a non-hydrolyzed or hydrolyzed succinimide or M¹ forms adilactam with B (e.g., a dilactam is formed when B reacts with thesuccinimide ring), wherein the succinimide or dilactam is conjugated tothe Ligand unit via a thioether linkage;

V, Q, T, and G are independently selected from —(C(R⁹)(R¹⁰))—;

R¹ is H or C₁-3 alkyl;

R⁹ and R¹⁰ are, in each occurrence, independently selected from H orC₁₋₃ alkyl;

F is C(E¹)(E²) wherein E¹ and E² are independently selected fromhydrogen, an electron withdrawing group, or E¹ and E² together are (═O);

RS is a reactive site for conjugation to a component of the optionalsecondary linker assembly or Drug unit;

g is 0 to 5;

m is 0 to 5;

n is 0 to 5;

d is 0 or 1;

x is 0 to 4, provided that when m is 0, x is 1 to 4;

and B is a base.

In some aspects, there are from 1 to 20 drug-linkers attached to eachLigand unit.

In selected embodiments, Ligand-Drug Conjugates have the formula:

or a pharmaceutically acceptable salt thereof.

In selected embodiments, Drug-linkers have the formula:

or a pharmaceutically acceptable salt thereof.

In selected embodiments, Linkers have the formula

or a pharmaceutically acceptable salt thereof.

In selected embodiments, Ligand-Linker Conjugates have the formula:

or a pharmaceutically acceptable salt thereof.

In formulas IIIa, IIIb, IIIc, and IIId or pharmaceutically acceptablesalts thereof:

-   -   L, if present, is a Ligand unit;    -   L^(O) is an optional secondary linker assembly;    -   RG, if present, is a reactive group (comprising a reactive site)        at the terminus of L^(O) which is suitable for attaching a Drug        unit;    -   M¹, if present, is a non-hydrolyzed or hydrolyzed succinimide or        M¹ forms a dilactam with B (e.g., a dilactam is formed when the        base reacts with the succinimide ring), wherein the succinimide        or dilactam is conjugated to the Ligand unit via a thioether        linkage;    -   V, Q, T, and G are independently selected from —(C(R⁹)(R¹⁰))—;        -   R¹ is H or C₁₋₃ alkyl;        -   R⁹ and R¹⁰ are, in each occurrence, independently selected            from H or C₁₋₃ alkyl;        -   F is C(E¹)(E²) wherein E¹ and E² are independently selected            from hydrogen, an electron withdrawing group, or E¹ and E²            together are (═O);        -   RS is a reactive site for conjugation to a component of the            optional secondary linker assembly or Drug unit;    -   g is 0 to 5;    -   m is 0 to 5;    -   n is 0 to 5;    -   d is 0 or 1;    -   x is 0 to 4, provided that when m is 0, x is 1 to 4;    -   p, if present, ranges from 1 to 20, preferably 1 to 12;    -   and B is a base.

It will be understood that for Formula III (including IIIa, IIIb, IIIc,and IIId) and pharmaceutically acceptable salts thereof, the electronwithdrawing group will either be represented by F (e.g., E¹, E² or E¹and E²) or by the reactive site RS. For example, when d is zero, or whenE¹ and E² are hydrogen, the reactive site will act as an electronwithdrawing group.

In some aspects, when d is zero, RS is —C(═O)—. In some aspects, n, d,and g are zero or m, n, d, and g are zero and RS is —C(═O)—.

Exemplary embodiments wherein the Ligand-Drug Conjugates, Drug-Linkers,Linkers, or Ligand-Linker conjugates are represented by Formula III (orformulae IIIa, IIIb, IIIc, or IIId, as the case may be) orpharmaceutically acceptable salts thereof, include those wherein m iszero; m is zero and n is zero, one, two, or three; x is 1; x is zero andn is zero, one, two, or three; and m is zero, n is zero, and x is 1.Exemplary embodiments include those described herein wherein R⁹ and R¹⁰are hydrogen. Exemplary embodiments include those wherein E¹ and E² areindependently selected from H, —CN, —NO₂, —CX₃ wherein X is halogen orE¹ and E² together are (═O). The remainder of the substituents are asdefined.

Exemplary embodiments wherein the Ligand-Drug Conjugates, Drug-Linkers,Linkers, or Ligand-Linker conjugates are represented by Formula III (orformulae IIIa, IIIb, IIIc, or IIId, as the case may be) orpharmaceutically acceptable salts thereof include those wherein:

-   -   (i) E¹ and E² are independently selected from hydrogen, —CN,        —NO₂, —CX₃, and —X wherein X is halogen or E¹ and E² together        are (═O);    -   (ii) m is zero and n is zero, one two or three;    -   (iii) x is 1;    -   (iv) x is 4;    -   (v) x is zero, and n is zero, one, two or three;    -   (vi) m is zero, n is zero, and x is 1;    -   (vii) d is 1 and g is 1 to 5;    -   (viii) d is 1 and g is 2 to 5;    -   (ix) n, d, and g are zero;    -   (x) m, n, d, and g are zero;    -   (xi) RS is —C(═O)—;    -   (xii) E¹ and E² are together (═O);    -   (xiii) B is

or —N(R³)(R⁴), wherein R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independentlyselected from H or C₁₋₆ alkyl and e is 0 to 4;

-   -   (xiv) B is —N(R³)(R⁴), wherein R³ and R⁴ are independently        selected from H or C₁₋₆ alkyl;    -   (xv) B is as in (xiii) or (xiv) and R³, R⁴, R⁵, R⁶, R⁷ and R⁸        are independently selected from H or C₁₋₃ alkyl;    -   (xvi) B is as in (xiii) or (xiv) and R³, R⁴, R⁵, R⁶, R⁷ and R⁸        are independently selected from H or methyl;    -   (xvii) B is as in (xiii) or (xiv) or (xvi) and R³ and R⁴ are        hydrogen;    -   (xviii) B is as in (xiii) or (xiv) or (xvi) and at least one of        R³ and R⁴ are hydrogen;    -   (xix) B is as in (xiii) or (xiv) or (xvi) and at least one of R³        and R⁴ is not hydrogen;    -   (xx) R¹, R₉, and R¹⁰ are independently selected from H or        methyl;    -   (xxi) R¹, R⁹, and R¹⁰ are hydrogen;    -   (xxii) R¹, R⁹, and R¹⁰ are independently selected from H or        methyl;    -   (xxiii) The cleavable unit is present;    -   (xxiv) The cleavable unit is present and has the formula        -   (AA-AA)₁₋₆-; wherein AA is at each occurrence independently            selected from an amino acid;    -   (xxv) The cleavable unit is present and conjugated directly to        the Drug unit;    -   (xxvi) The cleavable unit is present and conjugated directly to        the Drug unit via a cleavable peptide, disulfide, or hydrazone        bond;    -   (xxvii) The cleavable unit is present and the Spacer and        Stretcher unit are absent;    -   (xxviii) The Drug is an auristatin;    -   (xxix) M¹ is a hydrolyzed or non-hydrolyzed succinimide;    -   (xxx) p is about 4;    -   (xxxi) p is about 8;    -   (xxxii) the t1/2 of hydrolysis of the thio-substituted        succinimide of the Self-Stabilizing unit is from about 10        minutes to about 2.5 hours at pH 7.4 and 22° C.;    -   (xxxiii) the t1/2 of hydrolysis of the thio-substituted        succinimide of the Self-Stabilizing unit is from about 10        minutes to about 1 hour at pH 7.4 and 22° C.;    -   (xxxiv) the t1/2 of hydrolysis of the thio-substituted        succinimide of the Self-Stabilizing unit is from about 10        minutes to about 30 minutes at pH 7.4 and 22° C.;    -   (xxxv) the Ligand unit is an antibody;    -   (xxxvi) the Ligand unit is an antibody and is attached to the        Linker unit though a cysteine residue of an interchain        disulfide;    -   (xxxvii) the Ligand unit is a monoclonal antibody;        and any combinations or subcombinations of (i) through (xxxvii)        provided that the combinations or subcombinations do not        conflict with each other (e.g., xxx and xxxi conflict because p        cannot be both about 4 and about 8). For example, in selected        embodiments, m is zero, and n is zero, one, two, or three. In        other selected embodiments, m is zero, n is zero, and x is one.        In any of these selected embodiments, d can be one and g can be        from 1 to 5 or d can be one and g can be from 2 to 5. In any of        these embodiments, one or more of (i), (iii) or (xi)-(xxxvi) can        apply.

In each of the selected embodiments wherein the Ligand-Drug Conjugates,Drug-Linkers, Linkers, or Ligand-Linker conjugates are represented byFormula III (or formulae IIIa, IIIb, IIIc, or IIId, as the case may be)or pharmaceutically acceptable salts thereof, the optional secondarylinker assembly can be represented by the following formula:

wherein -A- is an optional Stretcher unit, the subscript a′ is 0 or 1;—W— is an optional Cleavable unit, the subscript w′ is 0 or 1; and —Y—is an optional Spacer unit, and the subscript y′ is 0 or 1.

Also included are selected embodiments wherein the Linker has theformula:

or a pharmaceutically acceptable salt thereof wherein V, T, B, R¹, Q, F,G, m, x, n, d, and g are as defined for formula III and RG is a reactivegroup comprising a reactive site, RS, for conjugation to the Drug unit Dwhen the secondary linker assembly is absent or to a component of thesecondary linker assembly wherein the secondary linker assemblysecondary linker has the following formula:

wherein -A- is an optional Stretcher unit, the subscript a′ is 0 or 1;—W— is an optional Cleavable unit, the subscript w′ is 0 or 1; and —Y—is an optional Spacer unit, and the subscript y′ is 0 or 1.

Further Embodiments of the Invention

Exemplary self-stabilizing linker assemblies prior to conjugation with aLigand and following conjugation and hydrolysis of the thio-substitutedsucccinimide which is formed upon conjugation are as follows:

or pharmaceutically acceptable salts thereof, wherein V, Q, T, m, n, x,and B, are as defined above for Formula III or any other selectedembodiment, c is from 1 to 4, and R¹¹ and R¹² are, at each occurrence,independently selected from H or C₁-C₆ alkyl. In an exemplary embodimentc is 1 or 4. The “S” of the hydrolyzed thio-succinimide represents asulfur atom of the Ligand (e.g., antibody). The wavy line indicateslinkage to the secondary linker assembly or Drug unit. In an exemplaryembodiment, the wavy line indicates linkage to the following secondarylinker assembly

wherein -A- is an optional Stretcher unit, the subscript a′ is 0 or 1;—W— is an optional Cleavable unit, the subscript w′ is 0 or 1; and —Y—is an optional Spacer unit, and the subscript y′ is 0 or 1.

In some aspects of the present invention, a self-stabilizing linkerassembly may undergo macro-cyclization to form a dilactam as followswherein R represents the remainder of the conjugate:

Secondary Linker Assembly

The optional secondary linker can comprise a variety of linking groups.In each of the embodiments provided herein, including the specificallyrecited embodiments, L^(O) can be present and have the formula:

-   -   wherein    -   -A- is an optional Stretcher unit, the subscript a′ is 0 or 1;    -   —W— is an optional Cleavable unit, the subscript w′ is 0 or 1;        and    -   —Y— is an optional Spacer unit, and the subscript y′ is 0 or 1;

The optional secondary linker assembly can be a releaseable linkerassembly, L^(R). In those embodiments, w is 1. In some other aspects,the optional secondary linker assembly is a non-releasable linkerassembly. In those embodiments w is 0 and release of drug is via a totalprotein degradation pathway (i.e., non-cleavable pathway).

The Ligand Unit

In some embodiments of the invention, a Ligand Unit is present. TheLigand unit (L-) is a targeting agent that specifically binds to atarget moiety. The Ligand can specifically bind to a cell component (aCell Binding Agent) or to other target molecules of interest. In someaspects, the Ligand unit acts to deliver the Drug unit to the particulartarget cell population with which the Ligand unit interacts. Ligandsinclude, but are not limited to, proteins, polypeptides and peptides.Suitable Ligand units include, for example, antibodies, e.g.,full-length antibodies and antigen binding fragments thereof,interferons, lymphokines, hormones, growth factors andcolony-stimulating factors, vitamins, nutrient-transport molecules (suchas, but not limited to, transferrin), or any other cell binding moleculeor substance. In some aspects, the ligand is a non-antibody proteintargeting agent. In some aspects, a Ligand-Functional Agent is providedwherein D′ is a Detection Unit or Stabilizing unit and the Ligand unitis a protein (e.g., a non-antibody protein).

In some aspects, a Ligand unit forms a bond with the maleimide of theSelf-Stabilizing Basic unit via a sulfhydryl group of the Ligand to forma thio-substituted succinimide. The sulfhydryl group can be present onthe Ligand in the Ligand's natural state, for example anaturally-occurring antibody, or can be introduced into the Ligand viachemical modification.

It has been observed for bioconjugates that the site of drug conjugationcan affect a number of parameters including ease of conjugation,drug-linker stability, effects on biophysical properties of theresulting bioconjugates, and in-vitro cytotoxicity. With respect todrug-linker stability, the site of conjugation of a drug-linker to aligand can affect the ability of the conjugated drug-linker to undergoan elimination reaction and for the drug linker to be transferred fromthe ligand of a bioconjugate to an alternative reactive thiol present inthe milieu of the bioconjugate, such as, for example, a reactive thiolin albumin, free cysteine, or glutathione when in plasma. Use of theSelf-Stabilizing Linkers of the present invention is particularlybeneficial when conjugated to thiol residues at sites that aresusceptible to the elimination reaction and subsequent transfer ofdrug-linker if non-self-stabilizing alkyl maleimides are used (e.g.,maleimido-caproyl drug linker). Such sites include, for example, theinterchain disulfides as well as select cysteine engineered sites. Useof the Self-Stabilizing Linkers of the present invention provides astable linkage and ability to attach multiple drugs to each Ligand unit.

In one aspect, the Ligand unit has one or more lysine residues that canbe chemically modified to introduce one or more sulfhydryl groups. Thereagents that can be used to modify lysines include, but are not limitedto, N-succinimidyl S-acetylthioacetate (SATA) and 2-Iminothiolanehydrochloride (Traut's Reagent).

In another embodiment, the Ligand unit can have one or more carbohydrategroups that can be chemically modified to have one or more sulfhydrylgroups.

In another embodiment, the sulfhydryl groups is be generated byreduction of the interchain disulfides. Accordingly, in someembodiments, the Linker unit is conjugated to a cysteine residue of thereduced interchain disulfides.

In another embodiment, the sulfhydryl group is chemically introducedinto the antibody, for example by introduction of a cysteine residue.Accordingly, in some embodiments, the Linker unit is conjugated to anintroduced cysteine residue.

Useful non-immunoreactive protein, polypeptide, or peptide Ligandsinclude, but are not limited to, transferrin, epidermal growth factors(“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derivedgrowth factor, IL-2, IL-6, transforming growth factors (“TGF”), such asTGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin andinsulin-like growth factors I and II, somatostatin, lectins andapoprotein from low density lipoprotein.

Particularly preferred ligands are antibodies. Useful polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of immunized animals.

Useful monoclonal antibodies are homogeneous populations of antibodiesto a particular antigenic determinant (e.g., a cancer cell antigen, aviral antigen, a microbial antigen, a protein, a peptide, acarbohydrate, a chemical, nucleic acid, or fragments thereof). Amonoclonal antibody (mAb) to an antigen-of-interest can be prepared byusing any technique known in the art which provides for the productionof antibody molecules by continuous cell lines in culture.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, or chimerichuman-mouse (or other species) monoclonal antibodies. The antibodiesinclude full-length antibodies and antigen binding fragments thereof.Human monoclonal antibodies may be made by any of numerous techniquesknown in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA.80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; and Olssonet al., 1982, Meth. Enzymol. 92:3-16).

The antibody can be a functionally active fragment, derivative or analogof an antibody that immunospecifically binds to target cells (e.g.,cancer cell antigens, viral antigens, or microbial antigens) or otherantibodies bound to tumor cells or matrix. In this regard, “functionallyactive” means that the fragment, derivative or analog is able to elicitanti-anti-idiotype antibodies that recognize the same antigen that theantibody from which the fragment, derivative or analog is derived.Specifically, in an exemplary embodiment the antigenicity of theidiotype of the immunoglobulin molecule can be enhanced by deletion offramework and CDR sequences that are C-terminal to the CDR sequence thatspecifically recognizes the antigen. To determine which CDR sequencesbind the antigen, synthetic peptides containing the CDR sequences can beused in binding assays with the antigen by any binding assay methodknown in the art (e.g., the BIA core assay) (See, e.g., Kabat et al.,1991, Sequences of Proteins of Immunological Interest, Fifth Edition,National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J.Immunology 125(3):961-969).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)₂ fragments, Fab fragments, Fvs, single chainantibodies, diabodies, tribodies, tetrabodies, scFv, scFv-FV, or anyother molecule with the same specificity as the antibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as for example,those having a variable region derived from a murine monoclonal andhuman immunoglobulin constant regions. (See, e.g., U.S. Pat. No.4,816,567; and U.S. Pat. No. 4,816,397, which are incorporated herein byreference in their entirety.) Humanized antibodies are antibodymolecules from non-human species having one or more complementaritydetermining regions (CDRs) from the non-human species and a frameworkregion from a human immunoglobulin molecule. (See, e.g., U.S. Pat. No.5,585,089, which is incorporated herein by reference in its entirety.)Such chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in International Publication No. WO 87/02671; European PatentPublication No. 0 184 187; European Patent Publication No. 0 171 496;European Patent Publication No. 0 173 494; International Publication No.WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Publication No.012 023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987,Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol.139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985,Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst.80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986,BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature321:552-525; Verhoeyan et al., 1988, Science 239:1534; and Beidler etal., 1988, J Immunol. 141:4053-4060; each of which is incorporatedherein by reference in its entirety.

Completely human antibodies are particularly desirable and can beproduced using transgenic mice that are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes.

Antibodies include analogs and derivatives that are either modified,i.e., by the covalent attachment of any type of molecule as long as suchcovalent attachment permits the antibody to retain its antigen bindingimmunospecificity. For example, but not by way of limitation,derivatives and analogs of the antibodies include those that have beenfurther modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular antibody unit orother protein, etc. Any of numerous chemical modifications can becarried out by known techniques including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis in thepresence of tunicamycin, etc. Additionally, the analog or derivative cancontain one or more unnatural amino acids.

Antibodies can have modifications (e.g., substitutions, deletions oradditions) in amino acid residues that interact with Fc receptors. Inparticular, antibodies can have modifications in amino acid residuesidentified as involved in the interaction between the anti-Fc domain andthe FcRn receptor (see, e.g., International Publication No. WO 97/34631,which is incorporated herein by reference in its entirety).

Antibodies immunospecific for a cancer cell antigen can be obtainedcommercially or produced by any method known to one of skill in the artsuch as, e.g., chemical synthesis or recombinant expression techniques.The nucleotide sequence encoding antibodies immunospecific for a cancercell antigen can be obtained, e.g., from the GenBank database or adatabase like it, the literature publications, or by routine cloning andsequencing.

In a specific embodiment, known antibodies for the treatment of cancercan be used. Antibodies immunospecific for a cancer cell antigen can beobtained commercially or produced by any method known to one of skill inthe art such as, e.g., recombinant expression techniques. The nucleotidesequence encoding antibodies immunospecific for a cancer cell antigencan be obtained, e.g., from the GenBank database or a database like it,the literature publications, or by routine cloning and sequencing.

In another specific embodiment, antibodies for the treatment of anautoimmune disease are used in accordance with the compositions andmethods of the invention. Antibodies immunospecific for an antigen of acell that is responsible for producing autoimmune antibodies can beobtained from any organization (e.g., a university scientist or acompany) or produced by any method known to one of skill in the art suchas, e.g., chemical synthesis or recombinant expression techniques. Inanother embodiment, useful antibodies are immunospecific for thetreatment of autoimmune diseases include, but are not limited to,anti-nuclear antibody; anti-ds DNA; Anti-ss DNA, anti-cardiolipinantibody IgM, IgG; anti-phospholipid antibody IgM, IgG; anti-SMantibody; anti-mitochondrial antibody; thyroid antibody; microsomalantibody; thyroglobulin antibody; anti-SCL-70 antibody; anti-Joantibody; anti-U₁RNP antibody; anti-La/SSB antibody; anti-SSA; anti-SSBantibody; anti-perital cells antibody; anti-histones antibody; anti-RNPantibody; C-ANCA antibody; P-ANCA antibody; anti-centromere antibody;Anti-Fibrillarin antibody and anti-GBM antibody.

In certain embodiments, useful antibodies can bind to a receptor or areceptor complex expressed on an activated lymphocyte. The receptor orreceptor complex can comprise an immunoglobulin gene superfamily member,a TNF receptor superfamily member, an integrin, a cytokine receptor, achemokine receptor, a maj or histocompatibility protein, a lectin, or acomplement control protein. Non-limiting examples of suitableimmunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD20,CD22, CD28, CD30, CD70, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS.Non-limiting examples of suitable TNF receptor superfamily members areCD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-R¹, TNFR-2, RANK,TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3,TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins areCD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d,CD49e, CD49f, CD103, and CD104. Non-limiting examples of suitablelectins are C-type, S-type, and I-type lectin.

The Drug Unit, D

The drug unit (D) can be any cytotoxic, cytostatic or immunosuppressivedrug also referred to herein as a cytotoxic, cytostatic orimmunosuppressive agent. The Drug unit has an atom that can form a bondwith the Linker Unit. In some embodiments, the Drug unit D has anitrogen atom that can form a bond with the Linker unit. In otherembodiments, the Drug unit D has a carboxylic acid that can form a bondwith the Linker unit. In other embodiments, the Drug unit D has asulfhydryl group that can form a bond with the Linker unit. In otherembodiments, the Drug unit D has a hydroxyl group or ketone that canform a bond with the Linker unit.

Useful classes of cytotoxic or immunosuppressive agents include, forexample, antitubulin agents, auristatins, DNA minor groove binders, DNAreplication inhibitors, alkylating agents (e.g., platinum complexes suchas cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, pre-forming compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, or the like. Particularly examples of useful classes ofcytotoxic agents include, for example, DNA minor groove binders, DNAalkylating agents, and tubulin inhibitors. Exemplary cytotoxic agentsinclude, for example, auristatins, camptothecins, duocarmycins,etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes,benzodiazepines (e.g., pyrrolo[1,4]benzodiazepines (PBDs),indolinobenzodiazepines, and oxazolidinobenzodiazepines) and vincaalkaloids. Select benzodiazepine containing drugs are described in WO2010/091150, WO 2012/112708, WO 2007/085930, and WO 2011/023883.

Individual cytotoxic or immunosuppressive agents include, for example,an androgen, anthramycin (AMC), asparaginase, 5-azacytidine,azathioprine, bleomycin, busulfan, buthionine sulfoximine,calicheamicin, camptothecin, carboplatin, carmustine (BSNU), CC-1065,chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine,cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin(formerly actinomycin), daunorubicin, decarbazine, docetaxel,doxorubicin, etoposide, an estrogen, 5-fluordeoxyuridine,5-fluorouracil, gemcitabine, gramicidin D, hydroxyurea, idarubicin,ifosfamide, irinotecan, lomustine (CCNU), maytansine, mechlorethamine,melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C,mitoxantrone, nitroimidazole, paclitaxel, palytoxin, plicamycin,procarbizine, rhizoxin, streptozotocin, tenoposide, 6-thioguanine,thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16 andVM-26.

In some typical embodiments, suitable cytotoxic agents include, forexample, DNA minor groove binders (e.g., enediynes and lexitropsins, aCBI compound; see also U.S. Pat. No. 6,130,237), duocarmycins (see U.S.Publication No. 20060024317), taxanes (e.g., paclitaxel and docetaxel),puromycins, vinca alkaloids, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,echinomycin, combretastatin, netropsin, epothilone A and B,estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide,eleutherobin, and mitoxantrone.

In some embodiments, the Drug unit is an anti-tubulin agent. Examples ofanti-tubulin agents include, but are not limited to, taxanes (e.g.,Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vincaalkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine).Other antitubulin agents include, for example, baccatin derivatives,taxane analogs (e.g., epothilone A and B), nocodazole, colchicine andcolcimid, estramustine, cryptophysins, cemadotin, maytansinoids,combretastatins, discodermolide, and eleutherobin.

In certain embodiments, the cytotoxic agent is maytansine or amaytansinoid, another group of anti-tubulin agents. (ImmunoGen, Inc.;see also Chari et al., 1992, Cancer Res. 52:127-131 and U.S. Pat. No.8,163,888).

In some embodiments, the Drug unit is an auristatin. Auristatinsinclude, but are not limited to, AE, AFP, AEB, AEVB, MMAF, and MMAE. Thesynthesis and structure of auristatins are described in U.S. PatentApplication Publication Nos. 2003-0083263, 2005-0238649 2005-0009751,2009-0111756, and 2011-0020343; International Patent Publication No. WO04/010957, International Patent Publication No. WO 02/088172, and U.S.Pat. Nos. 7,659,241 and 8,343,928; each of which is incorporated byreference in its entirety and for all purposes. Exemplary auristatins ofthe present invention bind tubulin and exert a cytotoxic or cytostaticeffect on the desired cell line.

Exemplary auristatin Drug units have the following formula or apharmaceutically acceptable salt thereof wherein the wavy line indicatessite of attachment to the Linker unit:

In some embodiments, the Drug is a benzodiazepine (includingbenzodiazepine containing drugs e.g., pyrrolo[1,4]benzodiazepines(PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines).

PBDs are of the general structure:

but can differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether(NH—CH(OMe)) at the N10-C11 position, which is the electrophilic centreresponsible for alkylating DNA. All of the known natural products havean (S)-configuration at the chiral C11a position which provides themwith a right-handed twist when viewed from the C ring towards the Aring. This gives them the appropriate three-dimensional shape forisohelicity with the minor groove of B-form DNA, leading to a snug fitat the binding site. The ability of PBDs to form an adduct in the minorgroove enables them to interfere with DNA processing, hence their use asantitumour agents. The biological activity of these molecules can bepotentiated by, for example, joining two PBD units together throughtheir C8/C′-hydroxyl functionalities via a flexible alkylene linker. ThePBD dimers are thought to form sequence-selective DNA lesions such asthe palindromic 5′-Pu-GATC-Py-3′ interstrand cross-link which is thoughtto be mainly responsible for their biological activity.

There are a number of different assays that can be used for determiningwhether a Ligand-Drug Conjugate exerts a cytostatic or cytotoxic effecton a cell line. In one example for determining whether a Ligand-DrugConjugate exerts a cytostatic or cytotoxic effect on a cell line, athymidine incorporation assay is used. For example, cells at a densityof 5,000 cells/well of a 96-well plated is cultured for a 72-hour periodand exposed to 0.5 jiCi of ³H-thymidine during the final 8 hours of the72-hour period, and the incorporation of ³H-thymidine into cells of theculture is measured in the presence and absence of Ligand-DrugConjugate. The Ligand-Drug Conjugate has a cytostatic or cytotoxiceffect on the cell line if the cells of the culture have reduced³H-thymidine incorporation compared to cells of the same cell linecultured under the same conditions but not contacted with theLigand-Drug Conjugate.

In another example, for determining whether a Ligand-Drug Conjugateexerts a cytostatic or cytotoxic effect on a cell line, cell viabilityis measured by determining in a cell the uptake of a dye such as neutralred, trypan blue, or ALAMAR™ blue (see, e.g., Page et al., 1993, Intl.J. of Oncology 3:473-476). In such an assay, the cells are incubated inmedia containing the dye, the cells are washed, and the remaining dye,reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytoxicity (Skehan et al., 1990, J. Nat'lCancer Inst. 82:1107-12). Preferred Ligand-Drug Conjugates include thosewith an IC₅₀ value (defined as the mAB concentration that gives 50% cellkill) of less than 1000 ng/ml, preferably less than 500 ng/ml, morepreferably less than 100 ng/ml, even most preferably less than 50 oreven less than 10 ng/ml on the cell line.

General procedures for linking a drug to linkers are known in the art.See, for example, U.S. Pat. Nos. 8,163,888, 7,659,241, 7,498,298, U.S.Publication No. US20110256157 and International Application Nos.WO2011023883, and WO2005112919.

M¹—the Succinimide

A non-hydrolyzed succinimide (also referred to herein as a succinimidering) conjugated to the Ligand unit via a thioether linkage can berepresented as follows wherein R represents the remainder of the Linkerunit optionally conjugated to a Drug unit, Detection unit or StabilizingUnit:

A hydrolyzed succinimide (also referred to herein as a hydrolyzedsuccinimide ring) conjugated to the Ligand unit via a thioether linkagecan be represented as one of its two positional isomers as followswherein R represents the remainder of the Linker unit optionallyconjugated to a Drug unit, Detection unit or Stabilizing Unit:

It will be understood for the non-hydrolyzed succinimides and hydrolyzedsuccinimide representations, there can be from 1 to 20, preferably 1 to12, 1 to 10 or 1 to 8 self-stabilizing linkers conjugated to eachLigand. In some aspects, there are from 1 to 20, preferably 1 to 12, 1to 10 or 1 to 8 drug-linkers conjugated to each Ligand. Additionally,for the conjugates described herein where a Ligand is not attached, thesuccinimide is in an unsaturated form as a maleimide (capable ofreactive with a thiol or the Ligand).

Basic Units

In Formula I, as well as the other formulae comprising aself-stabilizing linker (L^(SS)), the Basic unit (BU) can be essentiallyany base capable of facilitating a hydroxide ion (or water) attack tohydrolyze a nearby succinimide group. Accordingly, BU represents any“base” but is typically a group comprising a tethered amine or nitrogencontaining heterocycle; the amine or nitrogen containing heterocycleacting as the base of the Basic unit. Representative amines include—N(R³)(R⁴) wherein R³ and R⁴ are independently selected from H or C₁₋₆alkyl, preferably H or methyl,

wherein R⁵, R⁶, R⁷ and R⁸ are, at each occurrence, independentlyselected from hydrogen or C₁₋₆ alkyl, preferably H or methyl, and e is0-4. In the formulae above, the wavy line indicates the point ofattachment to a tethering group (typically an alkylene linker—(C(R⁹)(R¹⁰))_(x)— wherein the subscript x is an integer of from 0 to 6(or 1 to 6) provided that if x is 0 there are no less than 2 interveningatoms between the base of the Basic unit and the nitrogen atom of thesuccinimide (hydrolyzed or non-hydrolyzed) or dilactam and R⁹ and R¹⁰are independently selected from H or C₁₋₃ alkyl. In some aspects, thealkylene linker is —(CH₂)_(x)— wherein the subscript x is an integer offrom 0 to 6 (or 1 to 6) provided that if x is 0 there are no less than 2intervening atoms between the base of the Basic unit and the nitrogenatom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam. Thesubscript x is preferably 0 to 4, 1 to 4, or from 1 to 3, or from 2 to3, or from 2 to 4, but can also be 0, 1, 2, 3 or 4. Accordingly, theBasic unit, will in some embodiments, be selected from the groupconsisting of —(CH₂)_(x)NH₂, —(CH₂)_(x)NHR^(a), and —(CH₂)_(x)NR^(a) ₂,wherein x is an integer of from 0 to 4, 1 to 4, or from 1 to 3, or from2 to 3, or from 2 to 4, but can also be 0, 1, 2, 3 or 4, and each R^(a)is independently selected from the group consisting of C₁₋₆ alkyl andC₁₋₆ haloalkyl, or two R^(a) groups are combined with the nitrogen towhich they are attached to form an azetidinyl, pyrrolidinyl orpiperidinyl group. In some aspects, the base will be a nitrogenous base.

Hydrolysis Enhancers (HE) and Electron-Withdrawing Groups

The hydrolysis enhancers (HE) of Formula I, as well as the otherformulae comprising a self-stabilizing linker (L^(SS)), can beessentially any electron-withdrawing group capable of facilitating thehydrolysis of a nearby succinimide group. The hydrolysis is furtherfacilitated by the Basic unit (BU) assisting a hydroxide ion (or water)attack to hydrolyze a nearby succinimide group; or to render the nearbysuccinimide group more susceptible to hydrolysis. Accordingly, HE caninclude a functional group that draws electrons away from a reactioncenter. Exemplary electron withdrawing groups include, but are notlimited to, —C(═O), (═O), —CN, —NO₂, —CX₃, —X, —COOR, —CONR₂, —COR,—COX, —SO₂R, —SO₂OR, —SO₂NHR, —SO₂NR₂, —PO₃R₂, —P(O)(CH₃)NHR, NO, —NR₃₊,—CR═CR₂, and —C≡CR wherein X is F, Br, Cl, or I, and R is, at eachoccurrence, independently selected from the group consisting of hydrogenand C₁₋₆ alkyl. Exemplary electron withdrawing groups can also includearyl groups (e.g., phenyl) and certain heteroaryl groups (e.g.,pyridine). The term “electron withdrawing groups” includes aryls orheteroaryls further substituted with electron withdrawing groups.

In some embodiments, HE comprises a carbonyl, sulfonyl or phosphorylmoiety. In some embodiments, the hydrolysis enhancer (HE) is —CH₂C(O)—,—C(O)—, —C(O)CH₂—, —CH₂CH₂C(O)—, or —CH₂C(O)NH—.

In some embodiments wherein HE is directly linked to the secondarylinker assembly or Drug unit or Stability unit or Detection unit, HEwill comprise a reactive site suitable for attachment to the optionalsecondary linker assembly or Drug unit. In some aspects, the electronwithdrawing group will itself act as both the electron withdrawing groupand a reactive site for attachment to the optional secondary linkerassembly or Drug unit (e.g., —C(═O)—).

The Optional Secondary Linker Assembly (L^(O))

As noted above, the optional secondary linker assembly can berepresented by the formula:

wherein -A- is an optional Stretcher unit, the subscript a′ is 0 or 1;—W— is an optional Cleavable unit, the subscript w′ is 0 or 1; and —Y—is an optional Spacer unit, and the subscript y′ is 0 or 1. The wavyline adjacent to the optional Stretcher unit indicates the site ofattachment to the self-stabilizing linker assembly and the wavy lineadjacent to the optional Spacer unit indicates the site of attachment tothe Drug unit.

General methods of linking a Drug unit, a Detection unit, or a Stabilityunit to a Ligand unit are known in the art and linkers known in the artcan be adapted for use with a self-stabilizing linker assembly ormodified to include a basic component and/or electron withdrawing groupusing the teachings described herein. For example, auristatin andmaytansine ADCs are currently in clinical development for the treatmentof cancer.

Monomethyl auristatin E is conjugated through a protease cleavablepeptide linker to an antibody, monomethyl auristatin F is conjugateddirectly to an antibody through maleimidocaproic acid, DM1 is conjugatedthrough a disulfide or directly through the heterobifunctional SMCClinker, and DM4 is conjugated through a disulfide linker. These linkersystems can be adapted for use with a self-stabilizing linker assemblyor modified to include a basic component and/or electron withdrawinggroup using the teachings described herein and provide release of drugby a cleavable or non-cleavable system depending on the linker systemused. Disulfide, thioether, peptide, hydrazine, ester, or carbamatebonds are all examples of bonds that can be used to connect a Drug Unitto a Linker Unit. Stretcher units, Cleavable units, and Spacer units aredescribed in more detail below.

Also contemplated within the present invention are branched linkers.Accordingly, in one aspect, the Stretcher unit is designed in such a wayto allow branching within the Linker unit, e.g., the attachment of morethan one Drug unit or Detection unit or Stabilizing unit to eachself-stabilizing linker assembly, as represented by the followingformula:

wherein the wavy line indicates the site of attachment to theself-stabilizing linker assembly, —W— is an optional Cleavable unit, thesubscript w′ is 0 or 1; —Y— is an optional Spacer unit, the subscript y′is 0 or 1, u is from 2 to 20 (preferably from 2 to 10); A is a Stretcherunit, A′ is an optional Stretcher unit component at the terminus of A;and a′ is 0 or 1. Each A′, W, Y, and D′ can be the same or different.Each Cleavable unit can be attached to the Stretcher unit (either A orA′) through the same or different functional group on the Stretcherunit. In some aspects, D′ is a Drug unit D.

Exemplary Ligand-Functional Agent Conjugates or Ligand-Drug Conjugateshaving either branched or non-branched linkers have the followingformulae:

or a salt thereof (e.g., pharmaceutically acceptable salt), wherein eachof L, M¹, HE, BU, D′, and the subscripts p, a, b, m, q, and r have themeanings provided for Formula I and any of the selected embodiments forFormula I, D is a Drug unit, the circle represents a scaffold that canbe C₁₋₈ alkylene, C₁₋₈ heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀heteroarylene, and optionally comprises a reactive site suitable forattachment to A, W, Y or D′ (or D as the case may be); —W— is anoptional Cleavable unit, the subscript w′ is 0 or 1; —Y— is an optionalSpacer unit, the subscript y′ is 0 or 1, A is a Stretcher unit, A′ is anoptional Stretcher unit component at the terminus of A; a′ is 0 or 1;and u is from 1 to 20 (preferably from 1 to 10) wherein when u is from 2to 20 A is present and when u is 1, A can be present or absent. Each A′,W, Y, and D′ (or D as the case may be) can be the same or different.Each Cleavable unit can be attached to the Stretcher unit (either A orA′) through the same or different functional group on the Stretcherunit. In some aspects, w′ is 1. In some aspects, w′ is 1 and a′ is 0. Inaspects, wherein the linker isn't branched u is 1 and a′ is 0. In otheraspects, wherein the linker is branched, u is from 2 to 20 (preferablyfrom 2 to 10). In each of these selected embodiments, the circle canrepresent a scaffold that is C₁₋₈ alkylene or C₁₋₈ heteroalkylene(preferably C₁₋₄ alkylene or C₁₋₄ heteroalkylene) or C₁₋₃ alkylene orC₁₋₃ heteroalkylene. In some such aspects, the alkylene is straightchain or branched.

Ligand-Functional Agent Conjugates having either branched ornon-branched linkers can be represented by the following formulas:

or a salt thereof (e.g., pharmaceutically acceptable salt), wherein eachof L, M¹, HE, BU, D′, and the subscript p have the meanings provided forFormula I and any of the selected embodiments for Formula I, the circlerepresents a scaffold that can be C_(1-s) alkylene, C_(1-s)heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and optionallycomprises a reactive site suitable for attachment to A, W, Y or D′, —W—is an optional Cleavable unit, the subscript w′ is 0 or 1; —Y— is anoptional Spacer unit, the subscript y′ is 0 or 1, A is a Stretcher unit,A′ is an optional Stretcher unit component at the terminus of A; a′ is 0or 1; and u is from 1 to 20 (preferably from 1 to 10), wherein when u isfrom 2 to 20 A is present and when u is 1, A can be present or absent.Each A′, W, Y, and D′ can be the same or different. Each Cleavable unitcan be attached to the Stretcher unit (either A or A′) through the sameor different functional group on the Stretcher unit. In some aspects, w′is 1. In some aspects, w′ is 1 and a′ is 0. In aspects, wherein thelinker isn't branched u is 1, a′ is 0, and A can be present or absent.In other aspects, wherein the linker is branched u is from 2 to 20(preferably from 2 to 10).

Ligand-Drug Conjugates having either branched or non-branched linkerscan be represented by the following formulas:

or a salt thereof (e.g., pharmaceutically acceptable salt), wherein eachof L, M¹, HE, BU, and the subscript p have the meanings provided forFormula I and any of the selected embodiments for Formula I, D is a Drugunit, the circle represents a scaffold that can be C₁₋₈ alkylene, C₁₋₈heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and optionallycomprises a reactive site suitable for attachment to A, W, Y or D, —W—is an optional Cleavable unit, the subscript w′ is 0 or 1; —Y— is anoptional Spacer unit, the subscript y′ is 0 or 1, A is a Stretcher unit,A′ is an optional Stretcher unit component at the terminus of A; a′ is 0or 1; and u is from 1 to 20 (preferably from 1 to 10), wherein when u isfrom 2 to 20 A is present and when u is 1, A can be present or absent.Each A′, W, Y, and D can be the same or different. Each Cleavable unitcan be attached to the Stretcher unit (either A or A′) through the sameor different functional group on the Stretcher unit. In some aspects, w′is 1. In some aspects, w′ is 1 and a′ is 0. In aspects, wherein thelinker isn't branched u is 1, a′ is 0, and A can be present or absent.In other aspects, wherein the linker is branched u is from 2 to 20(preferably from 2 to 10).

Functional Agent-Linker Conjugates having either branched ornon-branched linkers can be represented by the following formulas:

or a salt thereof (e.g., pharmaceutically acceptable salt), wherein eachof HE, BU, D′, and the subscripts p, a, b, m, q, and r have the meaningsprovided for Formula I and any of the selected embodiments for FormulaI, the circle represents a scaffold that can be C₁₋₈ alkylene, C₁₋₈heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and optionallycomprises a reactive site suitable for attachment to A, W, Y or D′; W—is an optional Cleavable unit, the subscript w′ is 0 or 1; —Y— is anoptional Spacer unit, the subscript y′ is 0 or 1, A is a Stretcher unit,A′ is an optional Stretcher unit component at the terminus of A; a′ is 0or 1; and u is from 1 to 20 (preferably from 1 to 10, wherein when u isfrom 2 to 20, A is present and when u is 1, A can be present or absent.Each A′, W, Y, and D′ can be the same or different. Each Cleavable unitcan be attached to the Stretcher unit (either A or A′) through the sameor different functional group on the Stretcher unit. In some aspects, w′is 1. In some aspects, w′ is 1 and a′ is 0. In aspects, wherein thelinker isn't branched u is 1, a′ is 0, and A can be present or absent.In other aspects, wherein the linker is branched u is from 2 to 20(preferably from 2 to 10). In each of these selected embodiments, thecircle can represent a scaffold that is C₁₋₈ alkylene or C₁₋₈heteroalkylene (preferably C₁₋₄ alkylene or C₁₋₄ heteroalkylene) or C₁-3alkylene or C₁₋₃ heteroalkylene. In some such aspects, the alkylene isstraight chain or branched. In each of these selected embodiments, D′can be D.

In some aspects

is represented by:

In some such aspects, D′ is D.

Ligand-Linker Conjugates having either branched or non-branched linkerscan be represented by the following formulas:

or a salt thereof (e.g., pharmaceutically acceptable salt), wherein eachof L, M¹, HE, BU, and the subscripts p, a, b, m, q, and r have themeanings provided for Formula I and any of the selected embodiments forFormula I, the circle represents a scaffold that can be C₁₋₈ alkylene,C₁₋₈ heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, andoptionally comprises a reactive site suitable for attachment to A, W, Yor D; RG is a reactive group (comprising a reactive site) at theterminus of

which is suitable for attaching a Drug unit (or alternatively aDetection unit or a Stability unit), W— is an optional Cleavable unit,the subscript w′ is 0 or 1; —Y— is an optional Spacer unit, thesubscript y′ is 0 or 1, A is a Stretcher unit, A′ is an optionalStretcher unit component at the terminus of A; a′ is 0 or 1; and u isfrom 1 to 20 (preferably from 1 to 10) wherein when u is from 2 to 20, Ais present and when u is 1, A can be present or absent. Each A′, W, Y,and D can be the same or different. Each Cleavable unit can be attachedto the Stretcher unit (either A or A′) through the same or differentfunctional group on the Stretcher unit. In some aspects, w′ is 1. Insome aspects, w′ is 1 and a′ is 0. In aspects, wherein the linker isn'tbranched u is 1, a′ is 0, and A can be present or absent. In otheraspects, wherein the linker is branched u is from 2 to 20 (preferablyfrom 2 to 10). In each of these selected embodiments, the circle canrepresent a scaffold that is C₁₋₈ alkylene or C₁₋₈ heteroalkylene(preferably C₁₋₄ alkylene or C₁₋₄ heteroalkylene) or C₁₋₃ alkylene orC₁₋₃ heteroalkylene. In some such aspects, the alkylene is straightchain or branched.

In some aspects

is represented by:

Branched or non-branched Linkers can be represented by the followingformulas:

or a salt thereof (e.g., pharmaceutically acceptable salt), wherein eachof the scaffold, HE, BU, and the subscripts a, b, m, q, and r have themeanings provided for Formula I and any of the selected embodiments forFormula I, the circle represents a scaffold that can be C₁₋₈ alkylene,C₁₋₈ heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, andoptionally comprises a reactive site suitable for attachment to A, W, Yor D; RG is a reactive group (comprising a reactive site) at theterminus of

which is suitable for attaching a Drug unit (or alternatively aDetection unit or a Stabilizing unit), W— is an optional Cleavable unit,the subscript w′ is 0 or 1; —Y— is an optional Spacer unit, thesubscript y′ is 0 or 1, A is a Stretcher unit, A′ is an optionalStretcher unit component at the terminus of A; a′ is 0 or 1; and u isfrom 1 to 20 (preferably from 1 to 10), wherein when u is from 2 to 20,A is present and when u is 1, A can be present or absent. Each A′, W, Y,and D can be the same or different. Each Cleavable unit can be attachedto the Stretcher unit (either A or A′) through the same or differentfunctional group on the Stretcher unit. In some aspects, w′ is 1. Insome aspects, w′ is 1 and a′ is 0. In aspects, wherein the linker isn'tbranched u is 1, a′ is 0, and A can be present or absent. In otheraspects, wherein the linker is branched u is from 2 to 20 (preferablyfrom 2 to 10). In each of these selected embodiments, the circle canrepresent a scaffold that is C₁₋₈ alkylene or C₁₋₈ heteroalkylene(preferably C₁₋₄ alkylene or C₁₋₄ heteroalkylene) or C₁₋₃ alkylene orC₁₋₃ heteroalkylene. In some such aspects, the alkylene is straightchain or branched.

In some aspects

is represented by:

In some other aspects, exemplary Ligand-Drug Conjugates having eitherbranched or non-branched linkers have the following formulae:

or a pharmaceutically acceptable salt thereof wherein each of L, M¹, V,R¹, T, B, Q, F, G, and RS and the subscripts p, m, x, n, d, and g havethe meanings provided for Formula III and any of the selectedembodiments for Formula III, L is a Ligand unit, W— is an optionalCleavable unit, the subscript w′ is 0 or 1; —Y— is an optional Spacerunit, the subscript y′ is 0 or 1, A is a Stretcher unit, A′ is anoptional Stretcher unit component at the terminus of A; a′ is 0 or 1;and u is from 1 to 20 (preferably from 1 to 10) wherein when u is from 2to 20, A is present and when u is 1, A can be present or absent. EachA′, W, Y, and D can be the same or different. Each Cleavable unit can beattached to the Stretcher unit (either A or A′) through the same ordifferent functional group on the Stretcher unit. In some aspects, w′is 1. In some aspects, w′ is 1 and a′ is 0. In aspects, wherein thelinker isn't branched u is 1, a′ is 0, and A can be present or absent.In other aspects, wherein the linker is branched u is from 2 to 20(preferably from 2 to 10).

Stretcher units, Cleavable units, and Spacer units are described in moredetail below.

The Stretcher Unit

The Stretcher unit (-A-), when present, extends the framework of theLinker unit to provide more distance between the self-stabilizing linkerassembly and the Drug unit. A Stretcher unit is capable of linking theself-stabilizing linker assembly to the Cleavable unit when theCleavable unit is present, the self-stabilizing linker assembly to theSpacer unit when the Cleavable unit is absent but the Spacer unit ispresent and the self-stabilizing linker assembly to the Drug unit whenboth the Cleavable unit and the Spacer unit are absent. As described, aStretcher unit is capable of attaching to more than one Cleavable unit,Spacer unit, and/or Drug unit.

The Stretcher unit can also act to alter the physiochemical propertiesof the Drug-Linker depending on components of the Stretcher unit. Insome aspects, the Stretcher unit will be added in order to increase thesolubility of the Drug-Linker and will comprise one or multiplesolubility-enhancing groups such as ionic groups or water-solublepolymers. Water-soluble typically includes any segment or polymer thatis soluble in water at room temperature and includespoly(ethylene)glycol groups as well as other polymers such aspolyethyleneimines.

A Stretcher unit can comprise one or multiple stretcher groups.Exemplary stretcher groups include, for example, —NH—C₁-C₁₀ alkylene-,—NH—C₁-C₁₀ alkylene-NH—C(O)—C₁-C₁₀ alkylene-, —NH—C₁-C₁₀alkylene-C(O)—NH—C₁-C₁₀ alkylene-, —NH—(CH₂CH₂O)_(s)—,—NH—(CH₂CH₂O)_(s)—CH₂—, —NH—(CH₂CH₂NH)_(s)—(CH₂)_(s),—NH—(CH₂CH₂NH)_(s)—(CH₂)_(s)—NH—C(O)—(CH₂)_(s), —NH—(C₃-C₈ carbocyclo)-,—NH-(arylene-)-, and —NH—(C₃-C₈ heterocyclo-)-, wherein each s isindependently 1-10. A representative stretcher group having a carbonylgroup for linkage to the remainder of the Linker unit or the Drug unitis as follow:

wherein R¹³ is —C₁-C₁₀ alkylene-, —C₃-C₈carbocyclo-, -arylene-,—C₁-C₃₀heteroalkylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈carbocyclo)-,—(C₃-C₈carbocyclo)-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, —(C₃-C₈ heterocyclo)-C₁-C₁₀ alkylene-,—(CH₂CH₂O)₁₋₁₀(—CH₂)₁₋₃—, or —(CH₂CH₂NH)₁₋₁₀ (—CH₂)₁₋₃—. In someembodiments, R¹³ is —C₁-C₁₀ alkylene- or —C₁-C₃₀heteroalkylene-. In someembodiments, R¹³ is —C₁-C₁₀ alkylene-, —(CH₂CH₂O)₁₋₁₀(—CH₂)₁₋₃—, or—(CH₂CH₂NH)₁₋₁₀(—CH₂)₁₋₃—. In some embodiments, R¹³ is —C₁-C₁₀alkylene-polyethyleneglycol, or polyethyleneimine.

Non-cleavable drug release systems are known in the art and can beadapted for use with the self-stabilizing linker assemblies of thepresent invention as Stretcher units and/or Spacer units. Anon-cleavable linker in capable of linking a Drug unit to a Ligand in agenerally stable and covalent manner and is substantially resistant toacid-induced cleavage, light-induced cleavage, peptidase- oresterase-induced cleavage, and disulfide bond cleavage. Drug is releasedfrom Ligand Drug Conjugates containing non-cleavable linkers viaalternative mechanisms, such as proteolytic ligand degradation.

Cross-linking reagents that form non-cleavable linkers betweenmaytansinoid drugs and ligands are well known in the art and can adaptedfor use herein. Exemplary cross-linking reagants that form non-cleavablelinkers between the maytansinoid drugs and ligands comprise a maleimidoor haloacetyl-based moiety. They include N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC), κ-maleimidoundecanoicacid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidylester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-(.alpha.-maleimidoacetoxy)-succinimide ester [AMAS],succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl4-(p-maleimidophenyl)-butyrate (SMPB), andN-(p-maleimidophenyl)isocyanate (PMPI),N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyliodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl3-(bromoacetamido)propionate (SBAP). Additional Stretcher units for usein combination with the self-stabilizing linker assembly of the presentinvention can be found, for example, in U.S. Pat. No. 8,142,784,incorporated herein by reference in its entirety and for all purposes.

The Cleavable Unit

The Cleavable unit (—W—), when present, is capable of linking theself-stabilizing linker assembly to the Spacer unit when the Spacer unitis present or the self-stabilizing linker assembly to the Drug unit whenthe Spacer unit is absent. The linkage from the self-stabilizing linkerassembly to the Spacer unit or to the Drug unit can be directly from theself-stabilizing linker assembly when the Stretcher unit is absent orvia the Stretcher unit if the Stretcher unit is present.

In some embodiment, the Cleavable unit will be directly conjugated tothe self-stabilizing linker assembly on one end and to the Drug unit onthe other end. In other embodiments, the Cleavable unit will be directlyconjugated to the Stretcher unit on one end and to the Drug unit on theother end. In yet other embodiments, the Cleavable unit will be directlyconjugated to the Stretcher unit on one end and to the Spacer unit onthe other end. In even yet other embodiments, the Cleavable unit will bedirectly conjugated to the self-stabilizing linker assembly on one endand to the Spacer unit on the other end. Any of specifically describedself-stabilizing linker assemblies described herein can be used in theseembodiments.

The Cleavable unit is capable of forming a cleavable bond with a Drugunit or a Spacer unit. Reactive groups for forming cleavable bonds caninclude, for example, sulfhydryl groups to form disulfide bonds,aldehyde, ketone, or hydrazine groups to form hydrazone bonds,carboxylic or amino groups to form peptide bonds, and carboxylic orhydroxy groups to form ester bonds.

The nature of the Cleavable unit can vary widely. For example, cleavablelinkers include disulfide containing linkers that are cleavable throughdisulfide exchange, acid-labile linkers that are cleavable at acidic pH,and linkers that are cleavable by hydrolases, peptidases, esterases, andglucoronidases.

In some aspects, the structure and sequence of the Cleavable unit issuch that the unit is cleaved by the action of enzymes present at thetarget site. In other aspects, the Cleavable unit can be cleavable byother mechanisms. The Cleavable unit can comprise one or multiplecleavage sites.

In some embodiments, the Cleavable unit will comprise one amino acid orone or more sequences of amino acids. The Cleavable unit can comprise,for example, a monopeptide, a dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,decapeptide, undecapeptide or dodecapeptide unit.

Each amino acid can be natural or unnatural and/or a D- or L-isomerprovided of course that there is a cleavable bond. In some embodiments,the Cleavable unit will comprise only natural amino acids. In someaspects, the Cleavable unit will comprise 1 to 12 amino acids incontiguous sequence.

In some embodiments, each amino acid is independently selected from thegroup consisting of alanine, arginine, aspartic acid, asparagine,histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine,leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan,valine, cysteine, methionine, selenocysteine, ornithine, penicillamine,β-alanine, aminoalkanoic acid, aminoalkynoic acid, aminoalkanedioicacid, aminobenzoic acid, amino-heterocyclo-alkanoic acid,heterocyclo-carboxylic acid, citrulline, statine, diaminoalkanoic acid,and derivatives thereof. In some embodiments, each amino acid isindependently selected from the group consisting of alanine, arginine,aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine,phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine,proline, tryptophan, valine, cysteine, methionine, and selenocysteine.In some embodiments, each amino acid is independently selected from thegroup consisting of alanine, arginine, aspartic acid, asparagine,histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine,leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan,and valine. In some embodiments, each amino acid is selected from theproteinogenic or the non-proteinogenic amino acids.

In another embodiment, each amino acid is independently selected fromthe group consisting of the following L-(natural) amino acids: alanine,arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid,glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine,isoleucine, tryptophan and valine.

In another embodiment, each amino acid is independently selected fromthe group consisting of the following D-isomers of these natural aminoacids: alanine, arginine, aspartic acid, asparagine, histidine, glycine,glutamic acid, glutamine, phenylalanine, lysine, leucine, serine,tyrosine, threonine, isoleucine, tryptophan and valine.

In some embodiments, the bond between the Cleavable unit and the Drugunit can be enzymatically cleaved by one or more enzymes, including atumor-associated protease, to liberate the Drug unit (-D), which in oneembodiment is protonated in vivo upon release to provide a Drug (D).

Useful Cleavable units can be designed and optimized in theirselectivity for enzymatic cleavage by a particular enzyme, for example,a tumor-associated protease. In one embodiment, a linkage (or bond)between the Cleavable unit and the Drug unit or Spacer unit is thatwhich cleavage is catalyzed by cathepsin B, C and D, or a plasminprotease.

In certain embodiments, the Cleavable unit can comprise only naturalamino acids. In other embodiments, the Cleavable unit can comprise onlynon-natural amino acids. In some embodiments, the Cleavable unit cancomprise a natural amino acid linked to a non-natural amino acid. Insome embodiments, the Cleavable unit can comprise a natural amino acidlinked to a D-isomer of a natural amino acid.

An exemplary Cleavable unit is the dipeptide -Val-Cit-, -Phe-Lys- or-Val-Ala.

In some embodiments, the Cleavable unit will comprises a peptide andwill comprise from 1 to 12 amino acids. In some such embodiments, thepeptide will be conjugated directly to the Drug unit and the Spacer unitwill be absent. In some such embodiments, the Stretcher unit and Spacerunit will be absent. In one aspect, the peptide will be a dipeptide.

In some embodiments, the Cleavable unit —W_(w)— will be represented by-(-AA-)₁₋₁₂-, or (-AA-AA-)₁₋₆ wherein AA is at each occurrenceindependently selected from natural or non-natural amino acids. In oneaspect, AA is at each occurrence independently selected from naturalamino acids. One of skill in the art would appreciate that amino acidsare typically linked to the Drug unit or Spacer unit through functionalunits present in the amino acid, e.g., its carboxylic acid or aminotermini.

In some such aspects, the Ligand Drug Conjugates and Drug-Linkers arerepresented by the following formulae or salts thereof wherein L,L^(SS), L^(TT) A, a′, AA, Y, y′, D, and p are as defined in any of theembodiments described herein and f is an integer from 1 to 12:

It will be understood that although not reflected in the above formulae,such formulaes can be modified as taught herein to include branchedlinkers, i.e., multiple Drug units can be attached to eachself-stabilizing linker assembly.

In other aspects, the Cleavable unit will comprise a glucoronide unit,preferably 1 or 2 glucoronide units. In some such embodiments, theGlucuronide unit comprises a sugar moiety (Su) linked via a glycosidebond (—O′—) to a self-immolative Spacer:

-[Su-O′—Y]—

The glycosidic bond (—O′—) is typically a β-glucuronidase-cleavage site,such as a bond cleavable by human, lysosomal β-glucuronidase.

In some aspects, -[Su-O′—Y]— is represented by the following formula:

wherein Su is a Sugar moiety, —O′— represents a glycosidic bond; each Ris independently hydrogen, a halogen, —CN, or —NO₂; wherein the wavybond adjacent to the nitrogen atom indicates covalent attachment to theStretcher unit or to the Ligand and the wavy bond adjacent to the oxygenindicates covalent attachment to the Spacer unit or to the Drug unit. Anexemplary Linker unit comprising a glucoronide prior to conjugation toan antibody and post conjugation is as follows wherein the wavy lineindicates attachment to a Drug unit or Spacer unit and Ab represents anantibody and S is a sulfur atom of the antibody. It will be understoodthat more than one self-stabilizing assembly can be attached to eachantibody:

In some embodiments, the Cleavable unit itself will comprise a sulfuratom that is capable of forming a bond with a sulfur atom of a Spacerunit or Drug unit to form a disulfide or hindered disulfide. Cleavageoccurs between the two sulfur atoms of the disulfide. In some suchembodiments, one of the sulfur atoms is cleaved from the Drug unit and,provided there is no further release mechanism, the other sulfur atomremains attached to the Drug unit. A Linker unit comprising a Cleavableunit having a sulfur atom is capable of forming a bond with a sulfuratom of a Spacer unit or Drug unit to form a disulfide or hindereddisulfide

Exemplary linkers include, for example, the following Drug-Linkerwherein the wavy line indicates the site of attachment to the remainderof the Linker unit, D is a maytansinoid drug, and R_(a) and R_(b) areindependently selected from H or methyl.

A variety of disulfide linkers are known in the art and can adapted foruse in the present invention, including, for example, those that can beformed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate), SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),and SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate). (See, e.g.,Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., InImmunoconjugates: Antibody Conjugates in Radioimagery and Therapy ofCancer (C. W. Vogel ed., Oxford U. Press, 1987. See also U.S. Pat. No.4,880,935.)

In some embodiments, the cleavable linker is pH-sensitive and willcomprise, for example, an acid-labile linker that is hydrolyzable in thelysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone,cis-aconitic amide, orthoester, acetal, or ketal group) can be used.(See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchikand Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989,Biol. Chem. 264:14653-14661.) Such linkers are relatively stable underneutral pH conditions, such as those in the blood, but are unstable atbelow pH 5.5 or 5.0, the approximate pH of the lysosome.

In some embodiments, the Cleavable unit will be conjugated directly tothe Drug unit and the Spacer unit will be absent and the Cleavable unitwill be linked to the Drug unit via a cleavable peptide, disulfide, orhydrazone bond.

The Spacer Unit

The Spacer unit (—Y—), when present, links a Cleavable unit to the Drugunit or a Stretcher unit to the Drug unit or a self-stabilizing linkerassembly to a Drug unit. Like the Stretcher unit, the Spacer unit, whenpresent can act to extend the framework of the Linker unit. The Spacerunit can comprise multiple self-immolative or non-self immolativegroups. In some embodiments, the Spacer unit comprises one or moreself-immolative groups. In this context, the term “self-immolativegroup” refers to a bifunctional chemical moiety that is capable ofcovalently linking together two spaced chemical moieties into a normallystable tripartite molecule. It will spontaneously separate from thesecond chemical moiety if its bond to the first moiety is cleaved. Inother embodiments, the Spacer unit is not self-immolative. In theseembodiments, part or all of the Spacer unit remains attached to the Drugunit.

In some embodiments, —Y— is a self-immolative group and is linked to aCleavable unit via the methylene carbon atom of the self-immolativegroup, and linked connected directly to the Drug unit via a carbonate,carbamate or ether group.

In some embodiments, -Yy- is a p-aminobenzyl alcohol (PAB) unit whosephenylene portion is optionally substituted with —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -halogen, -nitro or -cyano.

In another embodiment, -Yy- can be a carbonate group. An unsubstitutedPAB unit is as follows:

Other examples of self-immolative groups include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (see, e.g., Hay et al.,1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho orpara-aminobenzylacetals. Spacers can be used that undergo cyclizationupon amide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (see, e.g., Rodrigues et al., 1995, ChemistryBiology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (see, e.g., Storm et al., 1972, J Amer.Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (see, e.g.,Amsberry et al., 1990, J Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at the a-position of glycine(see, e.g., Kingsbury et al., 1984, J. Med. Chem. 27:1447) are alsoexamples of self-immolative groups.

Other suitable Spacer units are disclosed in Published U.S. PatentApplication No. 2005-0238649, the disclosure of which is incorporated byreference herein.

Exemplary Stretcher units, Cleavable units, and Spacer units that can beused with the present compositions and methods are described in WO2004010957, WO 2007/038658, WO 2005/112919, U.S. Pat. Nos. 6,214,345,7,659,241, 7,498,298, 7,968,687, 8,163,888, and U.S. Publication No.2009-0111756, 2009-0018086, 2009-0274713, each of which is incorporatedherein by reference in its entirety and for all purposes.

In embodiments wherein the Conjugates are conjugated to a Stabilizingunit or a Detection unit in lieu of a Drug unit, the optional SecondaryLinker Assembly will typically be absent. In embodiments where theSecondary Linker Assembly is present, the Stretcher unit will generallybe present but the Cleavable unit and the Spacer unit will be absent.The Stretcher unit will extend the framework of the Linker unit toprovide more distance between the self-stabilizing assembly and theDetection unit or Stability unit. In such aspects, the Stretcher unit iscapable of linking the self-stabilizing linker assembly to the Detectionunit or the Stabilizing unit.

Drug Loading

The number of self-stabilizing linkers per Ligand is represented by p.In embodiments wherein the linkers are not branched, p represents thenumber of drug-linker molecules (or detection-linker or stability-linkermolecules) per Ligand (e.g., antibody). Depending on the context, p canrepresent the average number of self-stabilizing linkers per Ligand (orin embodiments where the linkers are not branched, the average number ofdrug-linker molecules (or detection-linker or stability-linkermolecules) per Ligand (e.g., antibody)). The variable p ranges from 1 to20, typically 1 to 12, 1 to 10 and is preferably from 1 to 8. In somepreferred embodiments, when p represents the average number ofself-stabilizing linkers per antibody, p ranges from about 2 to about 5.In some embodiments, p is about 2, about 4, or about 8. In somepreferred embodiments, when p represents the average number ofdrug-linker molecules per antibody, p ranges from about 2 to about 5. Insome embodiments, p is about 2, about 4, or about 8. The number of D′per self-stabilizing linkers is represented by u. u ranges from 1 to 10.

The average number of Drugs units per Ligand unit in a preparation froma conjugation reaction may be characterized by conventional means suchas mass spectroscopy, ELISA assay, HIC and HPLC. The quantitativedistribution of Drug-Linker-Ligand conjugates in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous Ligand-Drug Conjugates, where p is acertain value from Ligand-Drug Conjugate with other drug loadings may beachieved by means such as reverse phase HPLC or electrophoresis.

Self-Stabilizing Linker Assembly (L^(SS) or L^(TT)) and Rates ofHydrolysis

The Self-Stabilizing linker assembly links the Ligand unit to aStretcher unit if the Stretcher unit is present, links the Ligand unitto a Cleavable unit if the Stretcher unit is absent and a Cleavable unitis present, links the Ligand unit to a Spacer unit if the Stretcher unitand the Cleavable unit are absent and the Spacer unit is present, orlinks the Ligand Unit to D′ (e.g., a Drug Unit) if the Stretcher unit,Cleavable unit and Spacer unit are absent. In some embodiments, theStretcher unit, Cleavable unit, and Spacer unit will be absent and theself-stabilizing linker assembly will be conjugated directly to D′(e.g., a Drug Unit). In other embodiments, one or more of the Stretcherunit, Cleavable unit, and Spacer unit will be present.

The rate at which the thio-substituted succinimide of theSelf-Stabilizing Linker when part of a Ligand-Drug Conjugate undergoeshydrolysis can be quantified using the t1/2 of hydrolysis. t1/2 ofhydrolysis refers to the time taken for half of the compound of interestto hydrolyze, i.e., undergo a ring opening, under stated conditions(e.g., pH 7.4 and 22° C.). In some embodiments of the present invention,the t1/2 of hydrolysis of the thio-substituted succinimide of theSelf-Stabilizing unit is less than 4 hours, preferably less than 3hours, even more preferably, less than 2 hours, less than 1 hour, lessthan 45 minutes, less than 30 minutes, less than 15 minutes using thefollowing assay and stated conditions.

The hydrolysis reaction rates of maleimido drug linkers followingconjugation to antibody cysteines can be determined by massspectrometry, as the hydrolyzed product has a molecular weight 18daltons greater than the unhydrolyzed conjugate. Reduction of theinterchain disulfides of a human IgG1 creates a single reduced cysteineon the light chain and three reduced cysteines on the heavy chain. Theself-stabilizing maleimide drug-linker can then be conjugated to thereduced antibody at pH 7.4 and 22° C. and introduced to ahigh-resolution electrospray mass spectrometer via a reversed-phase HPLCcolumn which separates the conjugated light and heavy chains. The massesof the conjugated light and heavy chains can thus be measured, and thepeak intensities determined by standard mass spectrometry dataprocessing software (e.g., MassLynx). By performing a series ofinjections over time, the disappearance of the peak corresponding to themass of the original, unhydrolyzed conjugate and the appearance of thepeak corresponding to the mass of the hydrolyzed conjugate can bemonitored, the intensities of the peaks determined, and the percentageof hydrolyzed conjugate calculated at each timepoint. By plotting thehydrolysis percentage versus time, a curve is generated (e.g., usingPRISM) which can be fit to a standard equation for exponential phenomenawhich includes a parameter for t1/2.

In some aspects, the Self-Stabilizing Linker will be designed such thatthe maleimide component of the Self-Stabilizing Linker does notsubstantially undergo hydrolysis prior to conjugation to the Ligandunit.

In some embodiments of the present invention, the t1/2 of hydrolysis ofthe thio-substituted succinimide of the Self-Stabilizing Linker is fromabout 5 or about 10 minutes to about 24 hours, preferably from about 5or about 10 minutes to about 12 hours, more preferably from about 5 orabout 10 minutes to about 5 hours, more preferably from about 5 or about10 minutes to about 2.5 hours, even more preferably from about 5 orabout 10 minutes to about 1 hour, even more preferably from about 5 orabout 10 minutes to about 30 minutes, even more preferably from about 5or about 10 minutes to about 20 minutes, and even more preferably fromabout 10 minutes to about 15 minutes at a pH of about 7 to about 7.5(e.g., 7.4) and a temperature of about 22° C.

In some such embodiments wherein the t1/2 of hydrolysis is as statedabove, the hydrolysis goes to completion. Complete hydrolysis isconsidered to be achieved if 90% of the thio-substituted succinimidehydrolyzes. Preferably, 95% or greater, 96%, 97%, 98%, 99% or 100%hydrolysis will be achieved. In some embodiments, the hydrolysisreaction will compete with a dilactam formation and will not achievecompletion. In some such embodiments, at least 90% of the reactionproduct will be a combination of either a hydrolyzed thio-substitutedsuccinimide Ligand-Drug Conjugate or a thio-substituted dilactamLigand-Drug Conjugate. Preferably at least 95% or greater, 96%, 97%,98%, 99% or 100% of the reaction product will be a combination of eithera hydrolyzed thio-substituted Ligand-Drug Conjugate or athio-substituted dilactam Ligand-Drug Conjugate. The percentage ofhydrolysis can be calculated from the mass spectrometric data of theconjugate at the final timepoint by determining the intensity of thepeak corresponding to the mass of the original, unhydrolyzed conjugateand the intensity of the peak corresponding to the mass of thehydrolyzed conjugate, and using the sum of the peak intensities todetermine the percentage hydrolyzed and percentage unhydrolyzed.

In addition to characterizing the Ligand-Drug Conjugate by its t1/2 ofhydrolysis and/or the efficiency of the hydrolysis reaction, thestability of the Ligand-Drug Conjugate can be characterized by theability of the Ligand-Drug Conjugate to undergo an elimination reactionand for the Drug-Linker to be transferred from the Ligand unit to analternative reactive thiol present in the milieu of the Ligand-DrugConjugate. In some embodiments, the Drug-Linker will exhibit no orsubstantially no disassociation from the Ligand under the followingassay and stated conditions. The phrase “substantially no disassociationfrom the Ligand” is considered to be achieved if less than 40%,preferably less than 20%, even more preferably less than 10%, or evenmore preferably less than 5% or less than 2% of the Drug-Linker in asample disassociates from the Ligand.

The elimination of a drug-linker containing an enzyme-cleavable linkerfrom an antibody can be measured in ex vivo plasma by the followingmethod. The conjugate is placed in sterile plasma and incubated at 37°C. At the beginning of the incubation and at varying timepoints from 1hour to 1 week or longer, an aliquot is removed at frozen at −80° C.Upon completion of the timepoints, the samples are passed over a proteinA affinity resin to capture the antibody, the resin is washed withbuffer, and then drug is released from the captured antibody bytreatment with an appropriate enzyme (e.g. papain or proteinase K forpeptide-based cleavable linkers). The released drug can then bequantified by standard LC-MS methodology, and the quantity of drugmeasured at each timepoint divided by the quantity of drug measured forthe pre-incubation aliquot to determine the percentage of drug remainingconjugated to the antibody at each timepoint. The precision of thisassay can be improved by including an internal standard antibody-drugconjugate which is prepared using an isotopically labeled version of thesame drug-linker, such that the drug which is released from it can bedetected independently in the LC-MS assay from the drug released fromthe test drug-linker by virtue of its mass difference. This isotopicallylabeled internal standard antibody-drug conjugate is added to eachsample in equal amounts immediately prior to the protein A capture step.The quantitation of the drug released from the test ADC is thenperformed ratiometrically to the signal from the internal standard byconventional LC-MS techniques.

An alternative method for evaluating the elimination of a maleimidedrug-linker from an antibody (or other ligand) is to incubate theconjugate in buffer (e.g., phosphate-buffered saline) at slightlyelevated pH (e.g., pH 8.0) in the presence of a large excess of asmall-molecule thiol (e.g., N-acetyl cysteine, NAC) which will reactwith any maleimide that eliminates from the parent conjugate. LC-MSassays can be performed to detect and quantify the drug-linkerconjugated to NAC, or the parent ligand-conjugate. In the latter case,the ratio of the ligand-conjugate to unconjugated ligand can be measuredand will remain constant over time if the ligand-conjugate is stable.Additional methods are provided in the examples section.

Treatment of Cancer

The Ligand-Drug Conjugates are useful for inhibiting the multiplicationof a tumor cell or cancer cell, causing apoptosis in a tumor or cancercell, or for treating cancer in a patient. The Ligand-Drug Conjugatescan be used accordingly in a variety of settings for the treatment ofcancers. The Ligand-Drug Conjugates can be used to deliver a drug to atumor cell or cancer cell. Without being bound by theory, in oneembodiment, the Ligand unit of a Ligand-Drug Conjugate binds to orassociates with a cancer-cell or a tumor-cell-associated antigen, andthe Ligand-Drug Conjugate can be taken up (internalized) inside a tumorcell or cancer cell through receptor-mediated endocytosis or otherinternalization mechanism. The antigen can be attached to a tumor cellor cancer cell or can be an extracellular matrix protein associated withthe tumor cell or cancer cell. Once inside the cell, via a cleavable ornon-cleavable mechanism, depending upon the components of the linkersystem, the drug is released within the cell. In an alternativeembodiment, the Drug or Drug unit is cleaved from the Ligand-DrugConjugate outside the tumor cell or cancer cell, and the Drug or Drugunit subsequently penetrates the cell.

The Ligand-Drug Conjugates can provide conjugation-specific tumor orcancer drug targeting, thus reducing general toxicity of the drug. Insome embodiments, the Linker units stabilize the Ligand-Drug Conjugatesin blood, yet are capable of liberating drug once inside the cell.

In one embodiment, the Ligand unit binds to the tumor cell or cancercell.

In another embodiment, the Ligand unit binds to a tumor cell or cancercell antigen which is on the surface of the tumor cell or cancer cell.

In another embodiment, the Ligand unit binds to a tumor cell or cancercell antigen which is an extracellular matrix protein associated withthe tumor cell or cancer cell.

The specificity of the Ligand unit for a particular tumor cell or cancercell can be important for determining those tumors or cancers that aremost effectively treated. For example, a ligand drug conjugate having aBR96 Ligand unit can be useful for treating antigen positive carcinomasincluding those of the lung, breast, colon, ovaries, and pancreas.Ligand-Drug Conjugates having an anti-CD30 or an anti-CD70 bindingLigand unit can be useful for treating hematologic malignancies.

Other particular types of cancers that can be treated with a ligand drugconjugates include, but are not limited to, those disclosed in Table 1:

TABLE 1 Solid tumors, including but not limited to: fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer,kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovariancancer, prostate cancer, esophogeal cancer, stomach cancer, oral cancer,nasal cancer, throat cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, testicular cancer, small cell lung carcinoma, bladder carcinoma,lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, retinoblastoma blood-borne cancers,including but not limited to: acute lymphoblastic leukemia “ALL”, acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia “AML”, acute promyelocytic leukemia “APL”,acute monoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia “CML”, chronic lymphocytic leukemia “CLL”, hairycell leukemia, multiple myeloma acute and chronic leukemias:lymphoblastic, myelogenous, lymphocytic, myelocytic leukemias Lymphomas:Hodgkin's disease, non-Hodgkin's Lymphoma, Multiple myeloma,Waldenström's macroglobulinemia, Heavy chain disease, Polycythemia vera

Multi-Modality Therapy for Cancer

Cancers, including, but not limited to, a tumor, metastasis, or otherdisease or disorder characterized by uncontrolled cell growth, can betreated or inhibited by administration of a Ligand-Drug Conjugate.

In other embodiments, methods for treating cancer are provided,including administering to a patient in need thereof an effective amountof a Ligand-Drug Conjugate and a chemotherapeutic agent. In oneembodiment the chemotherapeutic agent is that with which treatment ofthe cancer has not been found to be refractory. In another embodiment,the chemotherapeutic agent is that with which the treatment of cancerhas been found to be refractory. The Ligand-Drug Conjugates can beadministered to a patient that has also undergone surgery as treatmentfor the cancer.

In some embodiments, the patient also receives an additional treatment,such as radiation therapy. In a specific embodiment, the Ligand-DrugConjugate is administered concurrently with the chemotherapeutic agentor with radiation therapy. In another specific embodiment, thechemotherapeutic agent or radiation therapy is administered prior orsubsequent to administration of a ligand drug conjugate.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the chemotherapeutic agents, such a standardof care chemotherapeutic agent(s), can be administered.

Additionally, methods of treatment of cancer with a Ligand-DrugConjugate are provided as an alternative to chemotherapy or radiationtherapy where the chemotherapy or the radiation therapy has proven orcan prove too toxic, e.g., results in unacceptable or unbearable sideeffects, for the subject being treated. The patient being treated can,optionally, be treated with another cancer treatment such as surgery,radiation therapy or chemotherapy, depending on which treatment is foundto be acceptable or bearable.

Treatment of Autoimmune Diseases

The Ligand-Drug Conjugates are useful for killing or inhibiting thereplication of a cell that produces an autoimmune disease or fortreating an autoimmune disease. The Ligand-Drug Conjugates can be usedaccordingly in a variety of settings for the treatment of an autoimmunedisease in a patient. The Ligand-Drug Conjugates can be used to delivera drug to a target cell.

Without being bound by theory, in one embodiment, the Ligand-DrugConjugate associates with an antigen on the surface of a target cell,and the ligand drug conjugate is then taken up inside a target-cellthrough receptor-mediated endocytosis. Once inside the cell, one or morespecific peptide sequences within the Linker unit are cleaved, resultingin release of the Drug or Drug unit. The released Drug or Drug unit isthen free to migrate in the cytosol and induce cytotoxic or cytostaticactivities. In an alternative embodiment, the Drug is cleaved from theLigand-Drug Conjugate outside the target cell, and the Drug or Drug unitsubsequently penetrates the cell.

In one embodiment, the Ligand unit binds to an autoimmune antigen. Inone aspect, the antigen is on the surface of a cell involved in anautoimmune condition.

In another embodiment, the Ligand unit binds to an autoimmune antigenwhich is on the surface of a cell.

In one embodiment, the Ligand unit binds to activated lymphocytes thatare associated with the autoimmune disease state.

In a further embodiment, the Ligand-Drug Conjugate kills or inhibit themultiplication of cells that produce an autoimmune antibody associatedwith a particular autoimmune disease.

Particular types of autoimmune diseases that can be treated with theligand drug conjugates include, but are not limited to, Th2 lymphocyterelated disorders (e.g., atopic dermatitis, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, and graft versus host disease); Th1 lymphocyte-relateddisorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis,Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primarybiliary cirrhosis, Wegener's granulomatosis, and tuberculosis);activated B lymphocyte-related disorders (e.g., systemic lupuserythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type Idiabetes); and those disclosed in Table 2.

TABLE 2 Active Chronic Hepatitis, Addison's Disease, AllergicAlveolitis, Allergic Reaction, Allergic Rhinitis, Alport's Syndrome,Anaphlaxis, Ankylosing Spondylitis, Anti-phosholipid Syndrome,Arthritis, Ascariasis, Aspergillosis, Atopic Allergy, AtropicDermatitis, Atropic Rhinitis, Behcet's Disease, Bird-Fancier's Lung,Bronchial Asthma, Caplan's Syndrome, Cardiomyopathy, Celiac Disease,Chagas' Disease, Chronic Glomerulonephritis, Cogan's Syndrome, ColdAgglutinin Disease, Congenital Rubella Infection, CREST Syndrome,Crohn's Disease, Cryoglobulinemia, Cushing's Syndrome, Dermatomyositis,Discoid Lupus, Dressler's Syndrome, Eaton-Lambert Syndrome, EchovirusInfection, Encephalomyelitis, Endocrine opthalmopathy, Epstein-BarrVirus Infection, Equine Heaves, Erythematosis, Evan's Syndrome, Felty'sSyndrome, Fibromyalgia, Fuch's Cyclitis, Gastric Atrophy,Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis,Goodpasture's Syndrome, Graft v. Host Disease, Graves' Disease,Guillain-Barre Disease, Hashimoto's Thyroiditis, Hemolytic Anemia,Henoch-Schonlein Purpura, Idiopathic Adrenal Atrophy, IdiopathicPulmonary Fibritis, IgA Nephropathy, Inflammatory Bowel Diseases,Insulin-dependent Diabetes Mellitus, Juvenile Arthritis, JuvenileDiabetes Mellitus (Type I), Lambert-Eaton Syndrome, Laminitis, LichenPlanus, Lupoid Hepatitis, Lupus, Lymphopenia, Meniere's Disease, MixedConnective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis,Pernicious Anemia, Polyglandular Syndromes, Presenile Dementia, PrimaryAgammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, PsoriaticArthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome,Schistosomiasis, Schmidt's Syndrome, Scleroderma, Shulman's Syndrome,Sjorgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, SystemicLupus Erythematosis, Takayasu's Arteritis, Temporal Arteritis,Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic EpidermalNecrolysis, Type B Insulin Resistance, Type I Diabetes Mellitus,Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom's Macroglobulemia,Wegener's Granulomatosis

Multi-Drug Therapy of Autoimmune Diseases

Methods for treating an autoimmune disease are also disclosed includingadministering to a patient in need thereof an effective amount of aLigand-Drug Conjugate and another therapeutic agent known for thetreatment of an autoimmune disease.

Treatment of Infectious Diseases

The Ligand-Drug Conjugates are useful for killing or inhibiting themultiplication of a cell that produces an infectious disease or fortreating an infectious disease. The Ligand-Drug Conjugates can be usedaccordingly in a variety of settings for the treatment of an infectiousdisease in a patient. The Ligand-Drug Conjugates can be used to delivera drug to a target cell. In one embodiment, the Ligand unit binds to theinfectious disease cell.

In one embodiment, the conjugates kill or inhibit the multiplication ofcells that produce a particular infectious disease.

Particular types of infectious diseases that can be treated with theLigand-Drug Conjugates include, but are not limited to, those disclosedin Table 3.

TABLE 3 Bacterial Diseases: Diphtheria, Pertussis, Occult Bacteremia,Urinary Tract Infection, Gastroenteritis, Cellulitis, Epiglottitis,Tracheitis, Adenoid Hypertrophy, Retropharyngeal Abcess, Impetigo,Ecthyma, Pneumonia, Endocarditis, Septic Arthritis, Pneumococca,Peritonitis, Bactermia, Meningitis, Acute Purulent Meningitis,Urethritis, Cervicitis, Proctitis, Pharyngitis, Salpingitis,Epididymitis, Gonorrhea, Syphilis, Listeriosis, Anthrax, Nocardiosis,Salmonella, Typhoid Fever, Dysentery, Conjunctivitis, Sinusitis,Brucellosis, Tullaremia, Cholera, Bubonic Plague, Tetanus, NecrotizingEnteritis, Actinomycosis, Mixed Anaerobic Infections, Syphilis,Relapsing Fever, Leptospirosis, Lyme Disease, Rat Bite Fever,Tuberculosis, Lymphadenitis, Leprosy, Chlamydia, Chlamydial Pneumonia,Trachoma, Inclusion Conjunctivitis Systemic Fungal Diseases:Histoplamosis, Coccidiodomycosis, Blastomycosis, Sporotrichosis,Cryptococcsis, Systemic Candidiasis, Aspergillosis, Mucormycosis,Mycetoma, Chromomycosis Rickettsial Diseases: Typhus, Rocky MountainSpotted Fever, Ehrlichiosis, Eastern Tick-Borne Rickettsioses,Rickettsialpox, Q Fever, Bartonellosis Parasitic Diseases: Malaria,Babesiosis, African Sleeping Sickness, Chagas' Disease, Leishmaniasis,Dum-Dum Fever, Toxoplasmosis, Meningoencephalitis, Keratitis,Entamebiasis, Giardiasis, Cryptosporidiasis, Isosporiasis,Cyclosporiasis, Microsporidiosis, Ascariasis, Whipworm Infection,Hookworm Infection, Threadworm Infection, Ocular Larva Migrans,Trichinosis, Guinea Worm Disease, Lymphatic Filariasis, Loiasis, RiverBlindness, Canine Heartworm Infection, Schistosomiasis, Swimmer's Itch,Oriental Lung Fluke, Oriental Liver Fluke, Fascioliasis,Fasciolopsiasis, Opisthorchiasis, Tapeworm Infections, Hydatid Disease,Alveolar Hydatid Disease Viral Diseases: Measles, Subacute sclerosingpanencephalitis, Common Cold, Mumps, Rubella, Roseola, Fifth Disease,Chickenpox, Respiratory syncytial virus infection, Croup, Bronchiolitis,Infectious Mononucleosis, Poliomyelitis, Herpangina, Hand-Foot-and-Mouth Disease, Bornholm Disease, Genital Herpes, Genital Warts,Aseptic Meningitis, Myocarditis, Pericarditis, Gastroenteritis, AcquiredImmunodeficiency Syndrome (AIDS), Human Immunodeficiency Virus (HIV),Reye's Syndrome, Kawasaki Syndrome, Influenza, Bronchitis, Viral“Walking” Pneumonia, Acute Febrile Respiratory Disease, Acutepharyngoconjunctival fever, Epidemic keratoconjunctivitis, HerpesSimplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Shingles,Cytomegalic Inclusion Disease, Rabies, Progressive MultifocalLeukoencephalopathy, Kuru, Fatal Familial Insomnia, Creutzfeldt-JakobDisease, Gerstmann-Straussler-Scheinker Disease, Tropical SpasticParaparesis, Western Equine Encephalitis, California Encephalitis, St.Louis Encephalitis, Yellow Fever, Dengue, Lymphocytic choriomeningitis,Lassa Fever, Hemorrhagic Fever, Hantvirus Pulmonary Syndrome, MarburgVirus Infections, Ebola Virus Infections, Smallpox

Multi-Drug Therapy of Infectious Diseases

Methods for treating an infectious disease are disclosed includingadministering to a patient in need thereof a Ligand-Drug Conjugate andanother therapeutic agent that is an anti-infectious disease agent.

Compositions and Methods of Administration

The present invention provides pharmaceutical compositions comprisingthe Ligand-Drug Conjugates described herein and a pharmaceuticallyacceptable carrier. The Ligand-Drug Conjugates can be in any form thatallows for the compound to be administered to a patient for treatment ofa disorder associated with expression of the antigen to which the Ligandunit binds. For example, the conjugates can be in the form of a liquidor solid. The preferred route of administration is parenteral.Parenteral administration includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques. In oneaspect, the compositions are administered parenterally. In one aspect,the compounds are administered intravenously.

The present invention also provides pharmaceutical compositionscomprising the Ligand-Functional Agent Conjugates described herein and apharmaceutically acceptable carrier. The Ligand-Drug Conjugates can bein any form that allows for the compound to be administered to a patientfor treatment of a disorder or for diagnostic purposes

Pharmaceutical compositions can be formulated so as to allow a compoundto be bioavailable upon administration of the composition to a patient.Compositions can take the form of one or more dosage units, where forexample, a tablet can be a single dosage unit.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the compound, the manner ofadministration, and the composition employed.

The composition can be, for example, in the form of a liquid. The liquidcan be useful for delivery by injection. In a composition foradministration by injection, one or more of a surfactant, preservative,wetting agent, dispersing agent, suspending agent, buffer, stabilizerand isotonic agent can also be included.

The liquid compositions, whether they are solutions, suspensions orother like form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or digylcerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as amino acids, acetates,citrates or phosphates; detergents, such as nonionic surfactants,polyols; and agents for the adjustment of tonicity such as sodiumchloride or dextrose. A parenteral composition can be enclosed inampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is an exemplaryadjuvant. An injectable composition is preferably sterile.

The amount of the conjugate that is effective in the treatment of aparticular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays can optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the compositions will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances.

The compositions comprise an effective amount of a compound such that asuitable dosage will be obtained. Typically, this amount is at leastabout 0.01% of a compound by weight of the composition.

For intravenous administration, the composition can comprise from about0.01 to about 100 mg of a Ligand-Drug Conjugate per kg of the animal'sbody weight. In one aspect, the composition can include from about 1 toabout 100 mg of a Ligand-Drug Conjugate per kg of the animal's bodyweight. In another aspect, the amount administered will be in the rangefrom about 0.1 to about 25 mg/kg of body weight of a compound.

Generally, the dosage of a compound administered to a patient istypically about 0.01 mg/kg to about 100 mg/kg of the subject's bodyweight. In some embodiments, the dosage administered to a patient isbetween about 0.01 mg/kg to about 15 mg/kg of the subject's body weight.In some embodiments, the dosage administered to a patient is betweenabout 0.1 mg/kg and about 15 mg/kg of the subject's body weight. In someembodiments, the dosage administered to a patient is between about 0.1mg/kg and about 20 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 0.1 mg/kg to about5 mg/kg or about 0.1 mg/kg to about 10 mg/kg of the subject's bodyweight. In some embodiments, the dosage administered is between about 1mg/kg to about 15 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 1 mg/kg to about10 mg/kg of the subject's body weight. In some embodiments, the dosageadministered is between about 0.1 to 4 mg/kg, even more preferably 0.1to 3.2 mg/kg, or even more preferably 0.1 to 2.7 mg/kg of the subject'sbody weight over a treatment cycle.

The Ligand-Functional Agent Conjugates e.g., Ligand-Drug Conjugates) canbe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa).Administration can be systemic or local. Various delivery systems areknown, e.g., encapsulation in liposomes, microparticles, microcapsules,capsules, and can be used to administer a compound. In certainembodiments, more than one compounds or composition is administered to apatient.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich a compound is administered. Such pharmaceutical carriers can beliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil. The carriers can be saline, gum acacia, gelatin, starchpaste, talc, keratin, colloidal silica, urea. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents can be used. Inone embodiment, when administered to a patient, the compound orcompositions and pharmaceutically acceptable carriers are sterile. Wateris an exemplary carrier when the compounds are administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol. The present compositions, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.

In an embodiment, the conjugates are formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to animals, particularly human beings.Typically, the carriers or vehicles for intravenous administration aresterile isotonic aqueous buffer solutions. Where necessary, thecompositions can also include a solubilizing agent. Compositions forintravenous administration can optionally comprise a local anestheticsuch as lignocaine to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where a conjugate isto be administered by infusion, it can be dispensed, for example, withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the conjugate is administered by injection, an ampoule ofsterile water for injection or saline can be provided so that theingredients can be mixed prior to administration.

The pharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Pharmaceutical compositions of the present invention comprise the LigandDrug Conjugates of the present invention and a pharmaceuticallyacceptable carrier. In some preferred embodiments, all, or substantiallyall, or more than 50% of the Ligand Drug Conjugates present in thepharmaceutical composition comprises a hydrolyzed thio-substitutedsuccinimide. In some preferred embodiments, more than 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%of the Ligand Drug Conjugates present in the pharmaceutical compositioncomprises a hydrolyzed thio-substituted succinimide.

Methods for Preparing Ligand-Drug Conjugates

In another aspect, the present invention provides methods of preparingLigand-Drug Conjugates or Ligand-Functional Agent Conjugates comprisinga Self-Stabilizing Linker.

In some embodiments, methods of the present invention comprise the stepsof providing a Drug-Linker or Linker unit as described herein,conjugating said Drug-Linker or Linker unit to a sulfhydryl group of aLigand unit to form a conjugate, allowing the resultant conjugate toundergo a hydrolysis reaction to form a Ligand-Drug conjugate comprisinga thio-substituted hydrolyzed succinimide.

The rate of the thio-substituted succinimide hydrolysis can bemanipulated by adjusting the reaction conditions following conjugationof the Drug-Linker to the Ligand, e.g., by adjusting the pH ortemperature. In some embodiments of the present invention, all,substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90% or even 95%of the thio-substituted succinimide is hydrolyzed without manipulationof the reaction conditions, i.e., the hydrolysis reaction occurs underthe same reaction conditions as the conjugation reaction. In someembodiments, all, substantially all, or at least 50%, 60%, 70%, 80%,85%, 90% or even 95% of the thio-substituted succinimide is hydrolyzedfrom 20 minutes, to 4 hours following conjugation, preferably from 20minutes to 2 hours following conjugation. In exemplary embodiments, theconjugation conditions are pH of about 7.4 and a temperature of about22° C.

In some embodiments, methods for preparing a Ligand-Drug Conjugatecomprises the steps of providing a Drug-Linker or Linker unit comprisinga Self-Stabilizing Linker; conjugating said Drug-Linker or Linker unitto a sulfhydryl group of a Ligand to form a Ligand-Drug Conjugateconjugate comprising a non-hydrolyzed thio-substituted succinimide;allowing the non-hydrolyzed thio-substituted succinimide to undergo ahydrolysis reaction, wherein all, substantially all, or at least 50%,60%, 70%, 80% or even 85% of the succinimide is hydrolyzed from 10minutes to 4 hours following conjugation. In some embodiments, all,substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90% or even 95%of the succinimide is hydrolyzed by 10 minutes, by 20 minutes, 40minutes 60 minutes, 90 minutes or 120 minutes following conjugation. Insome embodiments, the hydrolysis reaction occurs under the same reactionconditions as the conjugation reaction. In exemplary embodiments, theconjugation conditions are pH of about 7.4 and a temperature of about22° C.

Methods for Synthesizing Self-Stabilizing Linkers

The present invention provides, inter alia, Self-Stabilizing Linkers.Methods of preparing Self-Stabilizing Linker units are encompassedwithin the scope of the present invention.

Maleimide compounds are typically prepared from corresponding amines byreaction of the primary amine with maleic anhydride followed bycyclodehydration of the maleamic acid.

The overall scheme for the preparation of malemide compounds is shown inthe scheme below.

For preparation of maleimides containing basic groups in the side chainof the starting amine, such basic groups should be protected, ifnecessary. The appropriate protecting groups should be stable underconditions of maleimide preparation, yet should be removable later inthe presence of maleimide. Suitable protecting groups consist, but arenot limited to, acid labile protecting groups. “Boc” protecting group isone of the preferred protecting group.

The first step of maleimide preparation, the formation of the maleamicacid is very facile and can be usually accomplished in good yield byslow addition of the amine to a suspension containing a stoichiometricexcess of the maleic anhydride.

The second step, cyclodehydration of the maleamic acid, can beaccomplished in a number of ways known to skilled in the art. Forexample, the use of chemical dehydrating agents has been a wellestablished method for accomplishing this step. Carbodiimides incombination with isomerizing alcohols, for example: DCC/HOBt, have beenused to effect cyclodehydration of amic acids to maleimides.

Thermal cyclodehydration with use of azeotropic distillation in thepresence of acid catalyst is another well known method to generatemaleimides. The use of an azeotropic solvent permits the efficientremoval of the water co-product as it forms, thereby driving reaction tomaleimide. Suitable azeotropic solvents include cyclohexane, benzene,toluene, ethylbensene, mesitylene, and the like. Toluene is consideredto be the most desirable since it boils at 110° C. at atmosphericpressure. Boiling temperatures below 200° C. are preferable to minimizepossible thermal isomerization of maleamic acid to the morethermodynamically stable trans (fumaramic acid) structure.

The use of polar aprotic co-solvents can be beneficial for overall yieldimprovement as well as reducing time of cyclodehydration. Several polaraprotic solvents including dimethylformamide, dimethylacetamide,acetonitrile, N-methylpyrrolidone, dimethylsulfoxide, and sulfonate havebeen claimed to be useful. The most useful polar aprotic solvent isdimethylformamide.

Incorporation of certain amine salts instead of aprotic solvents can befurther beneficial for maleimide formation according to U.S. Pat. No.5,973,166.

One step microwave assisted maleimide synthesis has been also reportedstarting from maleic anhydride and an appropriate amine using no solvent(H. N. Borah, et al., J. Chem. Research (S), 1998, 272-272).

Example using water as a solvent for maleimide formation has beenreported in ARKIVOC, 2001 (v) 60-67 by V. Ondrus, et al.

Alternatively, maleimide compounds can be generated from maleimide andappropriate alcohol using, for example, Mitsunobu reaction conditions asshown in the scheme below (M. A. Walker, Tetrahedron Letters, 1994, v.35, n 5, pp. 665-668).

The self-stabilizing linker assembly of the present invention are linkedto the Stretcher unit, Cleavable unit, Spacer unit, or Drug unit usingthe teachings described herein in combination with methods known in theart. The Linkers and Drug-Linkers are conjugated to Ligand units usingteachings described herein in combination with methods known in the art.For example, for conjugation to interchain disulfides, an antibody canbe treated with a reducing agent, such as dithiothreitol (DTT) to reducesome or all of the interchain disulfide cysteine residues to form highlynucleophilic cysteine thiol groups. The full reduced antibody orpartially reduced antibody can be subsequently conjugated to themaleimide of the Linker Unit. In exemplary embodiments, conjugationconditions are gentle ones, pH of about 7 and a temperature of about 22°C.

Intermediates

The present invention provides intermediates for use in makingSelf-Stabilizing Linkers. Intermediates include the following wherein T,c, R¹¹ and R¹² are as previously described.

Mono-Thio-Substituted or Di-Thio-Substituted Maleimide or SuccinimideSelf-Stabilizing Linkers

In addition to designing self-stabilizing linkers for increasing thehydrolysis rates of mono thio-substituted succinimides, self-stabilizinglinkers can also be used to increase the hydrolysis rate ofmono-thio-substituted maleimides, di-thio-substituted maleimides, ordi-thio-substituted succinimides.

In view of the above, the present invention provides in one group ofembodiments, a Ligand-Functional Agent Conjugate comprising a Ligandunit and at least one Functional Agent selected from a Drug unit, aDetection Unit, or a Stabilizing Unit, wherein the Ligand unit and eachof the Functional Agent(s) are joined by a self-stabilizing linkerassembly comprising a succinimide ring, a maleimide ring, a hydrolyzedsuccinimide ring or a hydrolyzed maleimide ring wherein the succinimidering, maleimide ring, hydrolyzed succinimide ring or hydrolyzedmaleimide ring is directly conjugated to the Ligand unit via one or twothioether linkages; and a base and an electron withdrawing groupoperably linked to stabilize the conjugate in plasma relative to aLigand-Functional Agent Conjugate lacking the self-stabilizing linkerassembly (i.e. by increasing the rate of succinimide or maleimide ringhydrolysis). In some aspects, the electron withdrawing group ispositioned to increase the electrophilicity of the succinimide ormaleimide rendering it more reactive with water and the base ispositioned to assist the hydrolysis of the succinimide or maleimide ring(e.g., by an intramolecular base catalysis mechanism).

In some embodiments, the Ligand-Functional Agent Conjugates arerepresented by Formula IV or IVa:

or a salt thereof (e.g., pharmaceutically acceptable salt thereof);whereinL is a Ligand unitL_(L) is a Ligand unit that can be present or absent, wherein L andL_(L) can be the same or different Ligand units;D′ is a Drug unit, a Detection unit, or a Stabilizing unit;L^(O) is an optional secondary linker assembly;M² is a maleimide ring, a hydrolyzed maleimide, a succinimide ring, or ahydrolyzed succinimide conjugated to at least one of L or L_(L) via athioether linkage; and

-   -   BU is a Basic unit;    -   HE is a hydrolysis enhancer comprising an electron withdrawing        group;    -   The circle represents a scaffold that can be C₁₋₈ alkylene, C₁₋₈        heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and        optionally comprises a reactive site suitable for attachment to        L^(O), A, W, Y or D′;    -   the subscripts m, q and r are each 0 or 1, and the sum of m+q+r        is 0, 1 or 2 provided that if m+q+r is 0, the scaffold is a        C₆₋₁₀ arylene or C₄₋₁₀ heteroarylene;    -   the subscript p ranges from 1 to 20.        -   W— is an optional Cleavable unit,        -   the subscript w′ is 0 or 1;        -   —Y— is an optional Spacer unit,        -   the subscript y′ is 0 or 1,        -   A is an optional Stretcher unit,        -   A′ is an optional Stretcher unit component at the terminus            of A;        -   a′ is 0 or 1; and        -   u is from 1 to 20, with the proviso that when u is from 2 to            20, A is present and when u is 1, A can be present or            absent.

L, HE and BU, L^(O), A, W, and Y have the meanings provided for theLigand-Drug Conjugates. Additionally each of the specifically recitedselected embodiments for the circle, L, HE, BU, L^(O), A, W, and Y areequally applicable to these Conjugates. L and L_(L) can be differentLigand units or the same Ligand unit. In embodiments, wherein L andL_(L) are the same Ligand unit, the succinimide or maleimide can beconjugated to the Ligand unit on the same or different polypeptidechains of the Ligand unit.

In some aspects, when r is 1, HE does not comprise a carbonyl group,(i.e., C(═O))

In some aspects, m+q+r is 0, 1 or 2

In some aspects, r is zero.

In some aspects, the Ligand-Functional Agent Conjugate is represented byFormula IV or IVa or a salt thereof wherein r and m are zero and q isone.

In some aspects, the Ligand-Functional Agent Conjugate is represented byFormula IV or IVa or a salt thereof wherein the circle represents ascaffold that is C₁₋₈ alkylene or C₁₋₈ heteroalkylene (preferably C₁₋₄alkylene or C₁₋₄ heteroalkylene), r is zero, and the sum of m+q is 1. Insome such aspects, the scaffold is C₁₋₃ alkylene or C₁₋₃ heteroalkylene.In some such aspects, the alkylene is straight chain or branched.

In some aspects, the Ligand-Functional Agent Conjugate is represented byFormula IV or IVa or a salt thereof wherein the circle represents ascaffold that is C₁₋₈ alkylene or C₁₋₈ heteroalkylene (preferably C₁₋₄alkylene or C₁₋₄ heteroalkylene), and m and r are zero. In some suchaspects, the scaffold is C₁₋₃ alkylene or C₁₋₃ heteroalkylene. In somesuch aspects, the alkylene is straight chain or branched.

In some aspects, the Ligand-Functional Agent Conjugate is represented byFormula IV or IVa or a salt thereof wherein the circle represents ascaffold that is C₁, C₂, C₃ or C₄ straight or branched chain alkylene, ris zero, and the sum of m+q is 1.

In some aspects, the Ligand-Functional Agent Conjugate is represented byFormula IV or IVa or a salt thereof wherein the circle represents ascaffold that is C₁, C₂, C₃ or C₄ straight or branched chain alkylene,and m and r are zero.

In some aspects, m and r are zero and

is represented by:

In some aspects, there are no less than 2 and no more than 6 interveningatoms between the base of the Basic unit and the nitrogen atom of themaleimide or succinimide (hydrolyzed or non-hydrolyzed) and there are nomore than 5 atoms, no more than 4 atoms, no more than 3 atoms, or nomore than 2 intervening atoms between the electron withdrawing group andthe nitrogen atom of the maleimide or succinimide (hydrolyzed ornon-hydrolyzed).

In some aspects, M² is a succinimide ring or hydrolyzed succinimide andL_(L) is present. In some aspects, M² is a succinimide ring orhydrolyzed succinimide and L_(L) is absent. In some aspects, M² is amaleimide ring or hydrolyzed maleimide and L_(L) is present. In someaspects, M² is a maleimide ring or hydrolyzed maleimide and L_(L) isabsent. In each of these embodiments when L and L_(L) are present, L andL_(L) can be the same Ligand unit or different Ligand units. In someaspects wherein L and L_(L) are present and are the same Ligand unit,the maleimide or succinimide can be conjugated to the Ligand unit on thesame or different polypeptide chains of the Ligand unit.

In each of these embodiments, the alkylene or heteroalkylene chain canbe straight or branched. In some aspects, the alkylene or heteroalkylenechain will be a straight chain. In other aspects, it will be branched.

In each of these embodiments, p can range from 1 to 20, preferably 1 to12, even more 1 to 10 or 1 to 8.

In some aspects wherein the scaffold itself is directly linked to theoptional secondary linker assembly or D′, (for example, in selectembodiments when q is zero or when q is zero and r is zero), thescaffold will comprise a reactive site suitable for attachment to theoptional secondary linker assembly or D′

In some aspects wherein the scaffold itself is directly linked to theoptional secondary linker assembly or D′, (for example, in selectembodiments when q is zero or when q is zero and r is zero), thescaffold will comprise a reactive site suitable for attachment to A orD′.

The maleimide ring can be conjugated to the Ligand unit via one or twothioether linkages as illustrated below both in non-hydrolyzed andhydrolyzed form and the succinimide ring can be conjugated to the Ligandunit via two thioether linkages as illustrated below in bothnon-hydrolyzed and hydrolyzed form wherein the wavy line indicates thepoint of attachment to the remainder of the linker conjugate orlinker-functional agent conjugate:

In embodiments wherein the maleimide ring, hydrolyzed maleimide,succinimide ring, or hydrolyzed succinimide is conjugated to the Ligandvia two thioether linkages, p typically ranges from 1 to 10, or 1 to 8,or 1 to 4 and the maleimide or succinimide can be conjugated to the sameor different polypeptide chains of the Ligand. In some aspects, theLigand is an antibody. In other aspects, the Ligand is a non-antibodyprotein.

Functional Agent-Linker Conjugates having either branched ornon-branched linkers can be represented by the following formulas:

or a salt thereof (e.g., pharmaceutically acceptable salt thereof);whereinD′ is a Drug unit, a Detection unit, or a Stabilizing unit;L^(O) is an optional secondary linker assembly;Q and Z are hydrogen or halogen wherein at least one of Q and Z arehalogen;

-   -   BU is a Basic unit;    -   HE is a hydrolysis enhancer comprising an electron withdrawing        group;    -   the circle represents a scaffold that can be C₁₋₈ alkylene, C₁₋₈        heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and        optionally comprises a reactive site suitable for attachment to        L^(O), A, W, Y or D′;    -   the subscripts m, q and r are each 0 or 1, and the sum of m+q+r        is 0, 1 or 2 provided that if m+q+r is 0, the scaffold is a        C₆₋₁₀ arylene or C₄₋₁₀ heteroarylene;        -   W— is an optional Cleavable unit,        -   the subscript w′ is 0 or 1;        -   —Y— is an optional Spacer unit,        -   the subscript y′ is 0 or 1,        -   A is an optional Stretcher unit,        -   A′ is an optional Stretcher unit component at the terminus            of A;        -   a′ is 0 or 1; and        -   u is from 1 to 20, with the proviso that when u is from 2 to            20, A is present and when u is 1, A can be present or absent

In some aspects, the halogen is bromine.

HE and BU, L^(O), A, W, and Y have the meanings provided for theDrug-Linker Conjugates. Additionally each of the specifically recitedselected embodiments for the circle, HE, BU, L^(O), A, W, and Y areequally applicable to these Conjugates.

In some aspects, m and r are zero and

is represented by:

Ligand-Linker Conjugates having either branched or non-branched linkerscan be represented by the following formulas:

or a salt thereof (e.g., pharmaceutically acceptable salt thereof);whereinL is a Ligand unit;L_(L) is a Ligand unit that can be present or absent, wherein L andL_(L) can be the same or different Ligand units;RG is a reactive group (comprising a reactive site) at the terminusofL^(O) or

which is suitable for attaching a Drug unit, Detection unit orStabilizing unit;L^(O) is an optional secondary linker assembly that is present;M² is a maleimide ring, a hydrolyzed maleimide, a succinimide ring, or ahydrolyzed succinimide conjugated to at least one of L or L_(L) via athioether linkage; and

-   -   BU is a Basic unit;    -   HE is a hydrolysis enhancer comprising an electron withdrawing        group;    -   the circle represents a scaffold that can be C₁₋₈ alkylene, C₁₋₈        heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and        optionally comprises a reactive site suitable for attachment to        L^(O), A, W, Y or FA;    -   the subscripts m, q and r are each 0 or 1, and the sum of m+q+r        is 0, 1 or 2 provided that if m+q+r is 0, the scaffold is a        C₆₋₁₀ arylene or C₄₋₁₀ heteroarylene;    -   the subscript p ranges from 1 to 20.        -   W— is an optional Cleavable unit,        -   the subscript w′ is 0 or 1;        -   —Y— is an optional Spacer unit,        -   the subscript y′ is 0 or 1,        -   A is an optional Stretcher unit,        -   A′ is an optional Stretcher unit component at the terminus            of A;        -   a′ is 0 or 1; and        -   u is from 1 to 20, with the proviso that when u is from 2 to            20, A is present and when u is 1, A can be present or            absent.

L, HE and BU, L^(O), A, W, and Y have the meanings provided for theLigand-Drug Conjugates. Additionally each of the specifically recitedselected embodiments for the circle, L, HE, BU, L^(O), A, W, and Y areequally applicable to these Conjugates.

In some aspects, r and m are zero and

is represented by:

Branched or non-branched Linkers can be represented by the followingformulas:

or a salt thereof (e.g., pharmaceutically acceptable salt thereof);whereinRG is a reactive group (comprising a reactive site) at the terminus ofL^(O) or

which is suitable for attaching a Drug unit, Detection unit or Stabilityunit;L^(O) is an optional secondary linker assembly that is present;Q and Z are hydrogen or halogen wherein at least one of Q and Z arehalogen;

-   -   BU is a Basic unit;    -   HE is a hydrolysis enhancer comprising an electron withdrawing        group;    -   the circle represents a scaffold that can be C₁₋₈ alkylene, C₁₋₈        heteroalkylene, C₆₋₁₀ arylene, or C₄₋₁₀ heteroarylene, and        optionally comprises a reactive site suitable for attachment to        L^(O), A, W, Y or FA;    -   the subscripts m, q and r are each 0 or 1, and the sum of m+q+r        is 0, 1 or 2 provided that if m+q+r is 0, the scaffold is a        C₆₋₁₀ arylene or C₄₋₁₀ heteroarylene;        -   W— is an optional Cleavable unit,        -   the subscript w′ is 0 or 1;        -   —Y— is an optional Spacer unit,        -   the subscript y′ is 0 or 1,        -   A is an optional Stretcher unit,        -   A′ is an optional Stretcher unit component at the terminus            of A;        -   a′ is 0 or 1; and        -   u is from 1 to 20, with the proviso that when u is from 2 to            20, A is present and when u is 1, A can be present or            absent.

HE and BU, L^(O), A, W, and Y have the meanings provided for theLigand-Drug Conjugates. Additionally each of the specifically recitedselected embodiments for the circle, HE, BU, L^(O), A, W, and Y areequally applicable to these Conjugates.

In some aspects, r and m are zero and

is represented by:

Methods of preparing mono- or di-thio-substituted halomaleimides as wellas di-thiosubstituted succinimides are known in the art as are methodsof conjugating them to ligands, see for example, Ryan et al., Chem.Coomun., 2011, 47, 5452-5454 and Smith et al., J. Am. Chem. Soc. 2010,132(6), 1960-1965.

EXAMPLES Example 1—Synthesis of Representative Self-StabilizingComponents

In a 50 ml round bottom flask H-Lys(boc)-OH (246 mg, 1 mmol) and maleicanhydride (98 mg, 1 mmol) were dissolved in 1 ml (4 vol.) acetic acidand the solution was stirred at room temperature for 3 hours. Thereaction mixture was concentrated to an oil on the rotovap, and theproduct was precipitated by adding ˜10 ml dichloromethane. Theprecipitate was collected by vacuum filtration, washed withdichloromethane, and dried overnight in the vacuum oven. 270 mg ofproduct was recovered as a white powder (85% yield)

Maleyl-Lys(boc)-OH (100 mg, 0.29 mmol) was suspended in Toluene (3 ml)and triethylamine (224 uL) over molecular sieves in a 50 ml round bottomflask equipped with a condenser. DMA (˜150 uL) was added to aidsolubility. The solution was heated to 125° C. and refluxed for 4 hoursafter which the reaction was shown to be complete by LCMS. The reactionmixture was concentrated to dryness on the rotovap, redissolved in DMSOand purified by preparative HPLC. 56 mg of product was isolated as awhite powder. (60% yield)

Maleyl-DPR(boc)-OH was prepared in the same manner asMaleyl-Lysine(boc)OH. (503 mg, 67%)

Maleoyl-DPR(boc)-OH was prepared in the same manner as Maleoyl-Lys(boc).(340 mg, 71%)

Maleyl-dimethyllysine was prepared in the same manner asMaleyl-lys(boc)-OH with the exception that the product did notprecipitate after addition of dichloromethane. Instead the oil wasco-evaporated with 1:1 dichloromethane/hexane until a white foam wasobtained and dried under high vacuum overnight. (109 mg, 99%)

In a 10 ml round bottom flask, Maleyl-dimethyllysine (100 mg) wasdissolved in acetic acid (1 ml) and refluxed for 4 hours. After 4 hoursthe reaction mixture was concentrated to dryness on the rotovap anddried to a white foam under high vacuum. NMR of crude material shows˜80% conversion based on ratio of the singlet at 6.9 ppm and theolefinic protons from the starting material.

Example 2—Synthesis of mDPR-Val-Cit-PAB-MMAE

mDPR-Val-Cit-PAB-MMAE was prepared by coupling Boc-protected mDPR toVal-Cit-PAB-MMAE using standard methods for peptide coupling. The Bocgroup was removed in the final step.

Preparation of Fmoc-Val-Cit-PAB-MMAE

MMAE (5.34 g, 6.94 mmol), Fmoc-Val-Cit-PAB-OCO-pNP (5.0 g, 6.94 mmol)and HOBt (1.4 mmol) were charged to a 250 ml round bottom flask purgedwith N2 and dissolved in 15 ml of DMA. DIPEA (2.44 ml, 14 mmol) was thenadded, and the solution was stirred overnight at room temperature underinert atmosphere. The product was isolated by preparative HPLC, using alinear gradiant from 30% MeCN (0.05% TFA) to 100% MeCN (0.05% TFA) over40 min. Fractions containing product were concentrated on the rotovap toa white powder, affording 3.2 g (34%)

Preparation of Val-Cit-PAB-MMAE

A solution of 3.2 g of Fmoc-Val-Cit-PAB-MMAE in 7 ml DMF and 7 mldiethylamine was stirred for 3 hours at room temperature. The reactionmixture was then concentrated to a thick oil on the rotovap. The productwas precipitated in diethyl ether (100 ml) and filtered affording 2.0 gof product as an off white powder which was used without furtherpurification.

Preparation of mDPR (boc) Val-Cit-PAB-MMAE

In a 50 ml round bottom flask mDPR(boc)-OH (25 mg, 0.089 mmol),Val-Cit-PAB-MMAE (100 mg, 0.089 mmol), and HATU (41 mg, 0.107 mmol) weredissolved in 2 ml DMF. DIPEA (34 uL) was added and the solution wasstirred for 1 hr at rt. The reaction mixture was diluted with 1 ml DMSOand the product was isolated by preparative HPLC. (70 mg, 56%)

Preparation of mDPR-Val-Cit-PAB-MMAE

The above material was dissolved in 2 ml 10% TFA/dichloromethane andstirred for 1 h at rt. The reaction mixture was concentrated to dryness,reconstituted in 1 ml DMSO, and purified by preparative HPLC. (56 mg,86%)

Example 3—Monitoring Thiosuccinimide Hydrolysis

Thiosuccinimide hydrolysis of a self-stabilizing bioconjugate can bemonitored by electrospray mass spectrometry, since the addition of waterto the conjugate results in an increase of 18 Daltons to the observablemolecular weight of the conjugate. When a conjugate is prepared by fullyreducing the interchain disulfides of a human IgG1 antibody andconjugating the maleimide to the resulting cysteines, each light chainof the antibody will contain a single maleimide modification and eachheavy chain will contain three maleimide modifications (see FIG. 1,top). Upon complete hydrolysis of the resulting thiosuccinimides, themass of the light chain will therefore increase by 18 Daltons, while themass of the heavy chain will increase by 54 Daltons. This is illustratedin FIG. 1 (bottom), with the conjugation and subsequent hydrolysis of aself-stabilizing maleimide drug-linker of the present invention(mDPR-Val-Cit-PAB-MMAE, molecular weight 1289 Da) to the fully reducedanti-CD30 antibody cAC10. The presence of the single N-linkedglycosylation site on the heavy chain results in the heterogeneity ofmasses observed in the unconjugated antibody.

Example 4—Monitoring t1/2 Hydrolysis

By monitoring the intensities of the non-hydrolyzed and hydrolyzed peaksin the mass spectrum of a self-stabilizing bioconjugate over time(mDPR-Val-Cit-PAB-MMAE), the hydrolysis kinetics can be evaluated. Thisis done by plotting the percent of the total population which hashydrolyzed at each timepoint versus time (FIG. 2, top). These data arethen fit to the exponential equation

Y=Y_(max)×(1−e ^((−Kt)))

where Y is the observed percent hydrolysis at time t, Ymax is theasymptotic maximal % hydrolysis, and K is the hydrolysis rate constant.The half-life for the hydrolysis reaction is defined as

t _(1/2)=ln(2)/K

When this procedure is performed on the light chain of a reduced hIgG1antibody, the analysis is quite straightforward as there is only oneconjugation site per light chain and the reaction is a simpleprogression from the unhydrolyzed species to the hydrolyzed species witha mass change of 18 Daltons. Performing this analysis on the heavy chainis complicated by the fact that there are a total of three conjugationsites, resulting in a series of peaks of +18, +36, and +54 Daltons asthe conjugate undergoes hydrolysis. The analysis of the heavy chain isfurther complicated by the presence of multiple glycoforms. The analysispresented in FIG. 2 was performed by only evaluating the peaks arisingfrom the most abundant glycoform (the transition from 54195 Da to 54250Da) and assuming that these peaks are a reasonable surrogate for thewhole population of heavy chain glycoforms. As is evident in FIG. 2, theobserved kinetic profiles for light and heavy chains are very similar.For this reason, and because of the added complexities of quantifyinghydrolysis rates on the heavy chain noted above, most of the data tocharacterize hydrolysis rates of self-stabilizing maleimides conjugatedto antibodies was determined from evaluation of the light chainhydrolysis.

One limitation of this methodology is that the electrospray ionizationprocess tends to produce a small proportion of sodium adducts in theobserved peaks (approximately 10% under the conditions used to generatethe data in FIG. 2), which have an observed mass 22 Daltons greater thanthe parent mass. Many mass spectrometers do not have sufficiently highresolution to resolve this +22 mass from the +18 mass that results fromhydrolysis on a protein with a total molecular mass in excess of 25,000Daltons. Consequently, at the early timepoints when the degree ofhydrolysis is low, the appearance of a peak at approximately +20 Daltonsis a combination of these two effects which cannot be easily separatedexperimentally. As a result, the estimate of the percent of hydrolyzedproduct at the earliest timepoints is probably an overestimate, but themagnitude of this effect diminishes as the reaction proceeds towardcompletion

Example 5—Evaluating Spacing Between the Maleimide and Basic Group ofthe Self-Stabilizing Linker Assembly

It was hypothesized that the presence of a basic amino group adjacent tomaleimide would accelerate the hydrolysis of thiosuccinimides preparedwith those maleimides and thus result in stable bioconjugates. Thedistance between the maleimide and the basic amino group was recognizedas an important parameter in the design of such self-stabilizing units.To evaluate the role of this spacing, a series of maleimides wereprepared with the general structure

where x varied from 1 to 4. These maleimides were then conjugated to afully reduced human IgG1 at pH8 and 37° C. and immediately monitored byelectrospray mass spectrometry to determine the rate of hydrolysis. Thedistance between the basic group and the maleimide is inverselyproportional to the hydrolysis rate—that is, the greater the distance,the slower the hydrolysis. This result illustrates that positioning abasic amino group close to a maleimide results in an increase in therate of succinimide ring hydrolysis of bioconjugates prepared with themaleimide. However, even with the shortest spacing tested here (x=1), anantibody conjugate would have to be held at pH 8 and 37° C. forapproximately 5 hours to achieve complete hydrolysis (about 5half-lives). Exposure of an antibody or other protein to such conditionsfor extended periods can potentially result in covalent modificationsand misfolding events, and so maleimides with even faster hydrolysisrates were sought.

To prepare bioconjugates with faster hydrolysis rates, a series ofmaleimides were prepared with the general structure

where x=1 to 4 and R=val-cit-PAB-MMAE. These maleimides were thenconjugated to a fully reduced human IgG1 at pH8 and 37° C. andimmediately monitored by electrospray mass spectrometry to determine therate of hydrolysis. As shown in FIG. 4 (top), the distance between thebasic group and the maleimide within this series of structurally relatedcompounds exerts a profound influence on the progress of the hydrolysisreaction. As in the prior example, the shorter the distance between themaleimide and the basic amine, the faster the hydrolysis. Since basicconditions (i.e. high pH) are known to increase the rate of maleimideand succinimide ring hydrolysis, this effect is presumably an example ofintramolecular catalysis by a general base mechanism. Within this seriesthe compounds with x=2 and x=3 did not attain complete hydrolysis duringthe 3 hour incubation, instead reaching an asymptote at approximately80% and 50%, respectively (plots are normalized to the maximallyachieved hydrolysis). This phenomenon may arise from a competingreaction such as direct nucleophilic attack of the primary amine on thesuccinimide ring, or may be due to an isomeric impurity in the maleimidewhich leads to biphasic hydrolysis kinetics.

Example 6—Hydrolysis Kinetics

The previous examples illustrate the influence that a basic group canhave over the rate of succinimide ring hydrolysis in a bioconjugate,depending on the distance between the basic group and the maleimide ofthe parent molecule. However, it is expected that the presence ofelectron-withdrawing or -donating groups will also influence the rate ofring hydrolysis, since these groups will influence the electron density(and therefore electrophilicity) at the carbonyl carbons of the ring. Inthe conjugates of example 5, a carboxamide group is present in the alphaposition relative to the nitrogen of the ring (i.e. a single carbon atomis present between the nitrogen of the ring and the carbonyl carbon ofthe carboxamide). Because the carboxamide is a weak electron withdrawinggroup, its presence is likely to influence the observed hydrolysisrates. To better understand the relative contributions of the basicamino group and the electron-withdrawing carboxamide group on theobserved hydrolysis rates, a series of maleimides were conjugated to areduced human IgG1 antibody at pH 7.4, 22° C., and the hydrolysis ratesdetermined by mass spectrometry (FIG. 5). These maleimides containedjust the carboxamide in the alpha position (triangles), just the primaryamine in the beta position (inverted triangles), or both the carboxamideand the primary amine (circles). A control maleimide which containedneither group in proximity to the maleimide was also evaluated, althoughits hydrolysis is so slow that no reaction was observed under theseconditions and no data is plotted. Under these conditions theself-stabilizing maleimide which contains both the base and the electronwithdrawing group produced a bioconjugate with a hydrolysis t_(1/2) ofjust 12 minutes, while the maleimide containing only the amine yielded at_(1/2) of 2.5 hours, and the maleimide containing only the carboxamideyielded a t_(1/2) of 24 hours. This result indicates that the basicgroup and the electron withdrawing group act in concert to yield aconjugate with the very rapid hydrolysis kinetics which are mostconvenient for the manufacture of bioconjugates under the desirable mildconditions. Conjugates prepared with the diaminopropionyl maleimide(circles) exhibit ideal hydrolysis characteristics, with a t_(1/2) ofless than 15 minutes under very gentle conditions and the reactionapproaching 100% completion in about 2 hours.

Example 7—Evaluating Spacing Between the Maleimide and Carboxamide Groupof the Self-Stabilizing Linker Assembly

The rapid and complete succinimide hydrolysis observed in conjugatesprepared with self-stabilizing diaminopropionyl maleimido drug-linkersacid (DPR) shown in examples 5 and 6 above indicates the importance ofboth the basic group and the electron withdrawing group to the design. Asecond, isomeric maleimido drug-linker was prepared withdiaminopropionic acid to further evaluate the role of these twocomponents on the hydrolysis behavior of the resulting conjugates. Thestructures are termed α-maleimido DPR and β-maleimido DPR and are shownbelow.

Both DPR maleimides possess a basic primary amine which is separatedfrom the maleimido nitrogen by two carbon atoms. Both also possess anelectron withdrawing carboxamide group, however the distance from themaleimido nitrogen the carboxamide varies from 1 to 2 carbon units (αand β, respectively). Finally, the separation between the basic amineand the carboxamide also varies from 1 to 2 carbon units (β and α,respectively). Collectively, this means that in β-DPR the carboxamideexerts less electron withdrawing influence on the maleimide ring butmore electron withdrawing influence on the primary amine, relative toα-DPR. This is expected to slow the rate of hydrolysis by reducing boththe electrophilicity of the maleimide and the basicity of the primaryamine. When these maleimido drug-linkers were conjugated to reducedantibody and monitored for succinimide hydrolysis, a 17-fold lowerhydrolysis rate was observed for β-DPR relative to α-DPR (FIG. 6). Thisexample illustrates how the relative positioning of a basic group and anelectron withdrawing group can be used to ‘tune’ the hydrolysis rate.

Examples 8-15

To evaluate the stability and pharmacological activity of ADCs preparedwith self-stabilizing drug-linkers, a self-stabilizingmaleimido-drug-linker was prepared. This drug-linker contains themaleimido-DPR group coupled to the cytotoxic agent MMAE via aprotease-cleavable val-cit PAB self-immolative group (referred to hereinas maleimido-DPR-val-cit-PAB-MMAE). For comparison, a nonself-stabilizing drug-linker was used (referred to herein asmaleimido-caproyl-val-cit-PAB-MMAE). The only difference between theseagents is the unit between the maleimide and the valine group of theval-cit linker. The maleimide units of these drug-linkers can beprepared using maleic anhydride and mono-protected diaminopropionic acidand aminocaproic acid, respectively

Example 8—Evaluating Stability of Ligand-Drug Conjugates in Buffer

In standard buffer systems, maleimide elimination from a bioconjugateprepared using thiol-maleimide chemistry is essentially undetectablebecause the eliminated maleimide quickly reacts again with the thiol,resulting in an equilibrium which lies far toward the side of the intactconjugate. However, the addition of a thiol scavenger to the buffercreates a system in which maleimide that eliminates from thebioconjugate can instead react with the scavenger, resulting in apersistent, observable loss of the maleimide from the protein. Anexperiment using such a system was performed with antibody-drugconjugates prepared with a self-stabilizing diaminoproprionyl (DPR)maleimido drug-linker alongside a non-stabilizing caproyl drug-linker.ADCs were prepared with 8 drugs per antibody of eithermaleimido-DPR-val-cit-PAB-MMAE or maleimido-caproyl-val-cit-PAB-MMAEusing a fully reduced humanized IgG1. Drug loading was confirmed byreversed-phase HPLC on a polymeric PLRP-S column as described previously(Sun 2005). Complete succinimide hydrolysis of the self-stabilizinglinker was also confirmed by electrospray mass spectrometry. These ADCswere placed in 150 mM Tris buffer, pH 8, at 2.5 mg/mL, containing 10 mMN-acetylcysteine as a scavenger, and incubated for 2 weeks at 37° C. Atseven timepoints during the incubation, an aliquot of each ADC wasremoved and frozen at −80° C. Upon completion of the time-course, allsamples were analyzed by the above reversed-phase HPLC method todetermine the drug:antibody ratio. The results of this study are shownin FIG. 7. The ADC prepared with the self-stabilizing DPR maleimidodrug-linker exhibited minimal loss of drug over this timecourse (from8.0 to 7.9 drugs per antibody over 14 days), while the ADC prepared withthe caproyl maleimido drug-linker lost approximately half of its drugload (from 8.0 to 3.9 drugs per antibody over 14 days) under theseconditions.

Example 9—Ex Vivo Plasma Stability (Reversed Phase Method)

Assessing drug loading of humanized ADCs in non-human plasma samples bythe reversed-phase HPLC method described in Example 8 can be achieved byfirst isolating the ADCs with IgSelect resin (GE Healthcare), whichselectively binds to the human Fc domain. ADCs were prepared with 8drugs per antibody of either maleimido-DPR-val-cit-PAB-MMAE ormaleimido-caproyl-val-cit-PAB-MMAE using a fully reduced human IgG1.These ADCs (0.25 mg/mL) were incubated in sterile rat plasma for 7 daysat 37° C. At seven timepoints during the incubation, a 50 μL aliquot ofeach ADC was removed and frozen at −80° C. Upon completion of thetimecourse, ADCs were purified from each sample and analyzed byreversed-phase HPLC to determine the drug:antibody ratio. The results ofthis study are plotted in FIG. 8. As was observed in buffer, incubationof an ADC prepared with a self-stabilizing maleimide in rat plasma alsoresults in little or no observable loss of drug under conditions whichresult in the loss of approximately half of the drug from amaleimido-caproyl ADC.

Example 10—Ex Vivo Plasma Stability (Conjugated Drug Method)

A second assay format was utilized to assess ADC stability in rat andhuman plasma ex vivo. ADCs were prepared with 4 drugs per antibody ofeither maleimido-DPR-val-cit-PAB-MMAE ormaleimido-caproyl-val-cit-PAB-MMAE using a human IgG1 partially reducedto a level of 4 thiols per antibody (resulting in an ADC with 4 drugsper antibody). These two ADCs were spiked into rat and human plasma andincubated at 37° C. for 7 days. At seven timepoints during thisincubation, aliquots were removed and frozen at −80° C. until completionof the timecourse. ADCs were then isolated from each sample and MMAEreleased proteolytically from the isolated ADCs as described previously(Sanderson 2005). The released MMAE was then quantified by LC-MS/MS andnormalized to the initial value for each ADC (FIG. 9). In both rat andhuman plasma, the ADC prepared with a self-stabilizing maleimide lostlittle or no drug under these conditions, while approximately half ofthe drug was lost from a maleimido-caproyl ADC.

Example 11—In Vivo Stability

As described in Example 10 above, the drug:antibody ratio can bemeasured for ADCs in rat plasma by reversed-phase HPLC analysisfollowing purification with IgSelect resin. This method was applied tosamples derived from an in vivo pharmacokinetic experiment in rats. ADCswere prepared with 4 drugs per antibody of eithermaleimido-DPR-val-cit-PAB-MMAE or maleimido-caproyl-val-cit-PAB-MMAEusing a humanized IgG1 partially reduced to an average of 4 thiols perantibody (resulting in drug:antibody ratio of 4). These ADCs werefurther purified as described previously (Sanderson 2005) by hydrophobicinteraction chromatography to isolate the species containing 4 drugs perantibody. These ADCs were dosed intravenously at 10 mg/kg inSprague-Dawley rats. At five timepoints, three animals from each dosinggroup were sacrificed and the collected blood was processed to plasmaand frozen at −80° C. Upon completion of the study, all samples wereprocessed by the IgSelect resin method described above, except that thesample volume varied. The drug:antibody ratio at each timepoint in thisstudy are plotted in FIG. 10. As was observed in rat plasma ex vivo, theADC prepared with a self-stabilizing maleimide exhibits minimal loss ofdrug in vivo, dropping from an initial value of 4.1 drugs per antibodyto a value of 3.6 drugs per antibody (12% reduction) after 7 days.During this same timeframe, the drug:antibody ratio of an ADC preparedwith a maleimido-caproyl linker dropped from an initial value of 3.9 toa value of 1.5 (61% reduction). This illustrates that the increasedstability of a self-stabilizing drug-linker that is observed ex vivotranslates into an in vivo setting.

Example 12—Pharmacokinetics

Because maleimido-caproyl ADCs are prone to loss of drug throughmaleimide elimination whereas self-stabilizing maleimide ADCs are not,it is reasonable to predict that exposure to antibody-conjugated drugwill be greater following equivalent doses of the two ADCs. To confirmthis prediction, ADCs were prepared with 4 drugs per antibody of eithermaleimido-DPR-val-cit-PAB-MMAE or maleimido-caproyl-val-cit-PAB-MMAEusing a human IgG1 partially reduced to an average of 4 thiols perantibody (resulting in drug:antibody ratio of 4). These two ADCs weredosed at 2 mg/kg in Sprague-Dawley rats, and blood samples were taken atseven timepoints and processed to plasma. These plasma samples, alongwith standards of each ADC for the preparation of a calibration curve,were subj ected to the mAb Select resin capture and papain releaseprocedure described example 10 above to measure the concentration ofantibody-conjugated MMAE. Antibody-conjugated drug concentrations werehigher for the ADC prepared with the self-stabilizing drug-linker, withthe magnitude of the difference increasing with time (data not shown).Initial antibody-conjugated drug concentrations are superimposable,reflecting the equivalence of the dose and drug:antibody ratio of theADCs. However, divergence is observed within the first day, reaching atwo-fold difference by day 3. These higher concentrations resulted in anapproximately 40% greater antibody-conjugated drug AUC for theself-stabilized ADC relative to the maleimido-caproyl ADC

Example 13—Toxicology

To assess the impact of self-stabilizing maleimides on toxicology, ADCswere prepared with 4 drugs per antibody of eithermaleimido-DPR-val-cit-PAB-MMAE or maleimido-caproyl-val-cit-PAB-MMAEusing a humanized IgG1 (which has no known binding to any rat antigen)partially reduced to an average of 4 thiols per antibody (resulting inan average drug:antibody ratio of 4). These ADCs were dosedintravenously in female CD®IGS rats (Charles River Laboratories) at 10mg/kg (6 rats per test article plus 6 rats receiving vehicle only).Prior to dosing and at 3 post-dose timepoints, blood samples were takenfor hematology and serum chemistry analysis for biomarkers of toxicity.Neutropenia induced by the MMAE ADC appeared less severe for theself-stabilized conjugate than for the maleimido-caproyl ADC (data notshown).

Example 14—Plasma Concentration of Released Drug

The toxicology experiment described in example 13 above also includedblood draws at one hour and 24 hours post-dose, which along with the 4day and 7 day post-dose samples were analyzed for unconjugated MMAE inplasma by LC-MS/MS. The results of this analysis indicated that peakconcentrations of circulating MMAE are about 2-fold lower for theself-stabilizing maleimido-DPR-val-cit-PAB-MMAE ADC relative to themaleimido-caproyl-val-cit-PAB-MMAE (data not shown).

Example 15. Xenograft Activity

To evaluate the impact of self-stabilizing drug-linkers on the antitumoractivity of ADCs, conjugates were prepared with the anti-CD30 antibodycAC10 using drug-linkers containing the val-cit-PAB-MMAE cytotoxicpayload linked to the antibody via either a maleimido-caproyl group or aself-stabilizing maleimido-DPR group. These ADCs were evaluated in twoseparate murine xenograft models of CD30+ human malignancies. In thefirst model (FIG. 11), Karpas-299 (human ALCL) cells were implantedsubcutaneously in female SCID mice and tumors were allowed to grow avolume of approximately 250 mm³ before dosing at 1 mg/kg weekly forthree doses (six mice per dose group). All six mice dosed with themaleimido-caproyl ADC experienced some tumor growth delay relative tothe untreated group, and two animals experienced partial tumorshrinkage; however, all tumors grew out and the entire group waseuthanized with large tumors. The self-stabilizing ADC dose groupexperienced complete responses (no detectable tumor) in all six animals,with five animals experiencing durable regressions for the course of thestudy and only one animal sacrificed after its tumor had returned onstudy day 55. The results of this study indicate a significantly greaterin vivo antitumor activity for the ADC prepared with theself-stabilizing drug-linker. In the second model, L428 (human HodgkinLymphoma) cells were implanted subcutaneously in female NSG mice andtumors were allowed to grow a volume of approximately 100 mm³ beforedosing at 1 mg/kg every four days for four doses (six mice per dosegroup). All animals in both ADC dose groups experienced significantgrowth delay during treatment, however all tumors began growing outafter study day 28 with no significant difference betweenmaleimido-caproyl and self-stabilizing ADCs. Thus, the improvement inantitumor activity observed with the self-stabilizing ADC in xenograftstudies appears to be model-dependent.

What is claimed is:
 1. A method of treating cancer, an autoimmunedisease or an infectious disease that expresses a target antigencomprising administering a Ligand-Functional Agent Conjugate having theformula:

or a salt thereof, wherein L is a Ligand unit directly attached to thesuccinimide ring of the Functional Agent Conjugate via a thioetherlinkage, wherein the Ligand is a monoclonal antibody that specificallybinds to the target antigen; D′ is a Drug unit; the subscript p rangesfrom 1 to 20; —W— is an optional Cleavable unit, the subscript w′ is 0or 1; —Y— is an optional Spacer unit, the subscript y′ is 0 or 1; A′ isan optional Stretcher unit; and the subscript a′ is 0 or
 1. 2. Themethod of claim 1 wherein p is 1 to
 12. 3. The method of claim 1 whereinp is 1 to
 8. 4. The method of claim 1 wherein p is 1 to
 8. 5. The methodof claim 1 wherein D′ is a cytotoxic agent.
 6. The method of claim 1wherein D′ is an auristatin selected from the group consisting of AE,AFP, AEB, AEVB, MMAF and MMAE.
 7. The method of claim 1 wherein W_(w′)is selected from the group consisting of Val-Cit, Phe-Lys and Val-Ala.8. The method of claim 1 wherein Y_(y′) is a PAB unit.
 9. The method ofclaim 8 wherein the PAB unit has a structure of:

wherein the wavy line adjacent to the nitrogen atom indicates covalentbinding of that nitrogen atom to W and the # adjacent to the carbonylindicates covalent binding of its carbon atom to D′.
 10. The method ofclaim 1 wherein the subscript a′ is 0; W_(w′) is Val-Cit; and Y_(y′) isPAB.
 11. The method of claim 1 wherein the subscript a′ is 0; W_(w′) isVal-Cit; Y_(y′) is PAB; and D′ is a cytotoxic agent.
 12. The method ofclaim 1 wherein the subscript a′ is 0; W_(w′) is Val-Cit; Y_(y′) is PAB;D′ is a cytotoxic agent; and p is 1 to
 8. 13. The method of claim 1wherein D′ is MMAE.
 14. The method of claim 1 wherein the subscript a′is 0; W_(w), is Val-Cit; Y_(y′) is PAB; and D′ is MMAE.
 15. The methodof claim 1 wherein the Ligand-Functional Agent Conjugate has thestructure of:

or the structure wherein the succinimide ring is hydrolyzed, or a saltthereof, wherein mAb is a monoclonal antibody and S is a sulfur atomfrom the monoclonal antibody.
 16. The method of claim 15, wherein thesubscript p is about 4.